Immobilization and Stabilization of Acylase on Carboxylated

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Immobilization and Stabilization of Acylase on Carboxylated Polyaniline Nanofibers for Highly Effective Antifouling Application via Quorum Quenching Jeongjoon Lee, Inseon Lee, Jahyun Nam, Dong Soo Hwang, Kyung-Min Yeon, and Jungbae Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01528 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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ACS Applied Materials & Interfaces

Immobilization and Stabilization of Acylase on Carboxylated Polyaniline Nanofibers for Highly Effective Antifouling Application via Quorum Quenching

Jeongjoon Lee1, Inseon Lee1, Jahyun Nam1, Dong Soo Hwang2,3, Kyung-Min Yeon4,*, and Jungbae Kim1,5,*

1

Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Republic

of Korea 2

Division of Integrative Biosciences and Biotechnology, Pohang University of Science and

Technology, Pohang 37673, Republic of Korea 3

School of Environmental Science and Engineering, Pohang University of Science and

Technology, Pohang 37673, Republic of Korea 4

Construction Technology Team, Samsung C&T Corporation, Alpharium Bldg 1. 606-10,

Daewang pangyo-Ro, Seongnam-si, Gyunggi-Do 13530, Republic of Korea 5

Green School, Korea University, Seoul 02841, Republic of Korea

* Correspondence and requests for materials should be addressed to Dr. Kyung-Min Yeon (email: [email protected]) and Prof. Jungbae Kim (email: [email protected])

1 Environment ACS Paragon Plus


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ABSTRACT Acylase was immobilized and stabilized on carboxylated polyaniline nanofibers (cPANFs) for the development of antifouling nanobiocatalysis with high enzyme loading and stability. Acylase was immobilized via three different approaches: covalent attachment (CA), enzyme coating (EC), and magnetically separable enzyme precipitate coating (Mag-EPC). The enzyme activity per unit weight of cPANFs of Mag-EPC was 75 and 300 times higher than those of CA and EC, respectively, representing the improved enzyme loading in the form of Mag-EPC. After incubation under shaking at 200 rpm for 20 days, Mag-EPC maintained 55 % of initial activity, while CA and EC showed 3 and 16% of their initial activities, respectively. The antifouling of highly loaded and stable Mag-EPC against the biofilm/biofouling formation of Pseudomonas aeruginosa was tested under static and continuous flow conditions. The biofilm formation in the presence of 40 µg/mL Mag-EPC under static condition was 5 times lower than that of a control with no addition of Mg-EPC. Under continuous membrane filtration, Mag-EPC delayed the increase of trans-membrane pressure (TMP) more effectively as the concentration of added MagEPC was increased. When separating Mag-EPC and membranes in two different vessels under internal circulation of culture solution, Mag-EPC maintained higher permeability than the control of no Mag-EPC addition. It was also confirmed that the addition of Mag-EPC reduced the generation of N-acyl homoserine lactone (AHL) autoinducers. This result reveals that the inhibition of biofilm formation and biofouling in the presence of Mag-EPC is due to the hydrolysis of AHL autoinducers catalyzed by the immobilized and stabilized acylase in the form of Mag-EPC. Mag-EPC of acylase with high enzyme loadings and improved stability has demonstrated its great potential as an antifouling agent by reducing the biofilm formation and membrane biofouling based on ‘enzymatic quorum quenching’ of autoinducers.


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KEYWORDS: Biofilm, Antifouling, Acylase, Magnetically separable enzyme precipitate coating, Quorum quenching



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1. INTRODUCTION Biofilms are aggregated microorganisms, naturally occurring at any aqueous environment, which can adhere to a surface with slimy extracellular polymeric matrix.1-3 Biofilms can profoundly affect human health and industrial productivity. For example, biofilms can be a reservoir of pathogens and responsible for a wide variety of nosocomial infections through contamination of medical devices such as catheters, implants and wound dressings.4-5 Biofilm formation is an important issue in various industrial sectors, including water treatment, food/beverage processing and air ventilation.6-12 Generally, it is difficult to solve these biofilmrelated problems using conventional techniques, such as antibiotic treatment and chlorine injection, because bacterial biofilms have increased antibiotic resistance and mechanical strength compared to the planktonic cells.4,13 Quorum sensing represents the cell-to-cell communication via the signal molecules called autoinducers.14 These autoinducers control cell’s individual behaviors and make the cell population synchronize systematic and unified behaviors like a multicellular organism.15 As a result of quorum sensing, various physiological functions such as exoenzyme production, virulence factor secretion, bioluminescence and biofilm formation are regulated in a cell-density dependent way.16-18 Many studies indicate that quorum sensing is closely associated with the biofilm life-cycle of attachment, micro-colony, and maturation. Especially, quorum sensing plays a critical role in converting micro-colonies into a mature biofilm.19 Thus, the inactivation of quorum-sensing autoinducers using highly-selective enzyme reactions, called “enzymatic quorum quenching”, has been proposed as an effective strategy for the control of biofilm formation.20-22 Three types of auotoinducers have been well documented: cyclic peptides for Gram-positive, N-acyl homoserine lactone (AHL) for Gram-negative, and autoinducer-2 with structure of farnesyl borate dieter for both Gram positive and Gram-negative.23-25 Acylase is one 4 Environment ACS Paragon Plus

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of the well-known quorum quenching enzymes that can degrade the AHL autoinducers by cleaving the acyl amide bond between lactone ring and acyl chain. Previous studies have proposed the potential of antifouling using quorum quenching acylases.26-29 However, the short lifetime of enzymes themselves is one of the main problems against the successful antifouling application of enzymes under a long-term operation. Nanobiocatalysis, immobilizing and stabilizing enzymes using nanostructured materials, has demonstrated the potential solution for a long term operation of enzyme system with high enzyme loading and stability.30-31 For example, the approach of enzyme precipitation and crosslinking (EPC) has demonstrated its great potential in stabilizing the enzyme activity.32-33 Various enzymes such as glucose oxidase32 and carbonic anhydrase33 were immobilized via the approach of enzyme precipitate coating, which led to the improved enzyme loading and stability by adding the enzyme precipitation and crosslinking steps after the covalent enzyme attachment. In this study, we developed the highly effective antifouling system based on enzymatic quorum quenching (Figure 1). Acylase, as a quorum quenching enzyme, was immobilized on carboxylated polyaniline fibers (cPANFs) via three different approaches: covalent attachment (CA), enzyme coating (EC), and magnetically separable enzyme precipitate coating (Mag-EPC). cPANFs were selected for immobilization of acylase (AC) because they can be synthesized in a simple synthetic procedure and at a relatively low price, compared to other nanomaterials.33 Antifouling performance of acylase-immobilized cPANFs were tested using a model microorganism of Pseudomonas aeruginosa under various conditions such as static, singlevessel reactor, and two-vessel reactor.



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2. EXPERIMENTAL SECTION

2.1. Materials. Acylase (EC.3.5.1.14) from porcine kidney, N-acetyl-L-methionine, sodium phosphate monobasic, sodium phosphate dibasic, hydrochloric acid (HCl), Tris-HCl, Trizma base, glutaraldehyde, ammonium sulfate, 2-mercaptoethanol, aniline, 3-aminobenzoic acid, ethanol, acetonitrile, ammonium persulfate, o-phthaldialdehyde reagent solution incomplete, sodium tetraborate decahydrate, diethyl ether, dimethyl sulfoxide, hydroxyl amine, sodium hydroxide and ferric chloride were purchased from Sigma Aldrich (St. Louis, MO, USA). 1-Ethyl-3-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-(N-morpholino) ethanesulfonic acid (MES) were purchased from Pierce (Rockford, IL, USA). Amine functionalized magnetic nanoparticles (~50 nm diameter) were purchased from Chemicell (Berlin, Germany).

2.2. Synthesis of carboxylated polyaniline nanofibers. Carboxylated polyaniline nanofibers (cPANFs) were synthesized via copolymerization of aniline and 3-aminobenzoic acid (3-ABA) by following a protocol of the previous report.33 Briefly, aniline and 3-ABA under the molar ratio of 0.25:0.75 was added in 1 M HCl solution. To dissolve the aniline and 3-ABA, the mixed solution was shaken at 200 rpm and 50 °C for 1 h. Then, the same volume of 0.1 M ammonium persulfate in 1 M HCl solution was added to the solution of aniline and 3-ABA to initiate copolymerization. The mixed solution was shaken (200 rpm) at room temperature for 24 h. Then, cPANFs were washed with distilled water three times, and stored at 4 °C until use.


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2.3. Acylase immobilization on carboxylated polyaniline nanofibers. Acylase was immobilized onto cPANFs via the approach of magnetically separable enzyme precipitate coating (Mag-EPC), which consists of covalent attachment, ammonium sulfate enzyme precipitation and chemical cross-linking, together via two control approaches of covalent attachment (CA) and enzyme coating (EC) (Figure 2). cPANFs (2 mg) were first treated with EDC (10 mg/mL) and NHS (50 mg/mL) in MES buffer (100 mM, pH 6.5) under shaking at 50 rpm for 1 h. After washing with sodium phosphate buffer (100 mM, pH 7.0: PB), acylase in PB (1 mL of 30 mg/mL) was added in EDC/NHS-treated cPANFs, and the mixture was shaken at 150 rpm for 2 h to form the covalent bonds between acylase molecules and cPANFs for the synthesis of CA sample. To make the Mag-EPC sample, ammonium sulfate (final concentration of 10 % w/v) and amine-functionalized magnetic nanoparticles (2 mg) were added to the CA sample under shaking (200 rpm) for 30 min. Glutaraldehyde was added to make a final glutaraldehyde concentration of 0.5 % (w/v), and the mixture was shaken at 200 rpm for 30 min. To prepare the CA sample, 100 mM PB (pH 7.0) was added to the solution, instead of ammonium sulfate, magnetic nanoparticles and glutaraldehyde. For the preparation of the EC sample, PB was added to the solution, instead of ammonium sulfate and magnetic nanoparticles. Then, all the samples were incubated at 4 °C under shaking (50 rpm) for 12 h, and washed with PB. To cap the unreacted aldehyde groups, samples were treated with Tris-HCl buffer (100 mM, pH 7.0) under shaking (200 rpm) for 30 min. Then, samples were washed seven times with PB and stored in PB at 4 °C until use. For comparative studies of CA, EC and Mag-EPC, the amount of immobilized acylase samples is represented by the weight of cPANFs to be used for their immobilization.



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2.4. Amino acid analysis for the estimation of enzyme loadings and leachings. The enzyme loadings and leachings were estimated by quantifying the amino acid amount after enzymes were fully hydrolyzed.34 In details, immobilized samples or solutions with leached enzymes were hydrolyzed in 6 N HCl solution with 5% (v/v) phenol as an antioxidant at 156 °C and under argon for 1 h. The hydrolyzed samples were evaporated at 60 °C under vacuum, and then subsequently washed twice with distilled water and methanol, respectively. The dried samples were re-suspended in 300 µL of ninhydrin solution for the reaction with amine groups, and the amino acid analysis was performed using a ninhydrin-based amino acid analyzer (SYKAM System S4300, Eresing, Germany). The standard amino acids for the enzyme quantification were alanine, glycine, leucine and isoleucine.

2.5. Scanning electron microscope analysis. Scanning electron microscope (SEM) was performed to examine the morphology of cPANFs, CA, EC and Mag-EPC samples. All the samples were excessively washed with distilled water and freeze-dried for 24 h. After the platinum coating, the samples were analysed using a Quanta FEG 250 (FEI, Hillsboro, OR, USA) at an accelerating voltage of 15 kV.

2.6. Activity and stability measurements of acylase on cPANFs. The acylase activity was measured by a fluorometric method based on the reaction of ophthalaldehyde with the amino group of L-methionine, which is generated from the acylasecatalyzed hydrolysis of N-acetyl-L-methionine.28 The acylase-immobilized cPANFs were mixed with N-acetyl-L-methionine solution for an initiation of enzyme reaction. Then, 0.2 mL of aliquot was taken from the reaction solution at appropriate time intervals, and added to the 2.8 mL of o-


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phthalaldehyde solution (0.05 mg/mL) in 50 mM sodium borate buffer (pH 9.5). Upon excitation at 340 nm, the emission intensity at 455 nm was measured using a spectrofluorophotometer (RF5301, Shimadzu, Tokyo, Japan). The activity was calculated from the linear slope of timedependent increase of the emission intensity at 455 nm. One unit of acylase activity is defined by the hydrolysis for 1.0 µmole of N-acetyl-L-methionine per hour at pH 7.0 at room temperature. Kinetic constants (kcat and KM) were obtained from the nonlinear regression of kinetic analysis using a software (Enzyme Kinetics Pro from ChemSW, Fairfield, CA, USA). For stability measurement, the acylase-immobilized samples were incubated in PB (100 mM, pH 7.0) under shaking at 200 rpm. The residual activity of each sample was measured by using an aliquot from the stock solution after different times of incubation, and the relative activity was calculated by the ratio of residual activity at each time point to the initial activity of each sample.

2.7. Microplate assay for the biofilm quantification. Biofilm formation under different acylase dosing was evaluated by the microplate assay.35-36 Pseudomonas aeruginosa PAO1 was grown in Luria-Bertani (LB) broth (Difco, Franklin Lakes, NJ, USA) at 37 °C overnight. After bacterial cell was settled down through centrifugation (1000 rpm, 10 min), the pellet was re-suspended in the same volume of fresh LB medium. Then, 100 µL of P. aeruginosa culture and 60 µL of samples (Mag-EPC or cPANFs) were dispensed into a 96-well polystyrene microplate (Corning, Corning, NY, USA). The plate contents were incubated at 30 °C under static condition for 24 h for the biofilm growth. After the removal of the supernatant, each well was washed three times with distilled water and dried for 1 h. Then, biofilm in each well was stained with 0.01 % (w/v) crystal violet for 20 min. After discarding the staining solution, wells were washed three times with deionized water. The crystal violet bound



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to biofilm was solubilized in 200 µL of ethanol under shaking (100 rpm) for 3 min. Finally, the absorbance at 590 nm was measured using a microplate reader (Infinite 200 PRO, TECAN, Hombrechtikon, Switzerland) for biofilm quantification. All the experiments were performed with the triplicate samples.

2.8. One vessel system under continuous membrane filtration. Figure 3A shows the schematic of one vessel system for continuous membrane filtration. Briefly, polyvinylidene fluoride (PVDF) disk filter with 0.45 µm pore size (Merckmilipore, Billerica, MA, USA) was placed at the bottom of a stirred vessel (Amicon Model 8400, Merckmilipore, Billerica, MA, USA) with a working volume of 10 mL. After the reactor was filled with the overnight-culture solution of P. aeruginosa, synthetic wastewater and Mag-EPC (0, 10, 20, and 30 µg/mL) were added to the reactor. Then, synthetic wastewater was continuously fed to the reactor at a flow rate of 0.25 mL/min, and the membrane filtration was conducted at the same flow rate of 0.25 mL/min in order to maintain the constant working volume. The impeller speed was set to 100 rpm. During this continuous operation, the transmembrane pressure (TMP) was measured by using the digital pressure gauge (ZSE40A, SMC, Tokyo, Japan) in order to evaluate the biofouling on the membrane surface. The continuous membrane filtration was also performed under the recycle of filtrate and with no influent of synthetic water, which allows for the recycle of autoinducers in the filtrate and can potentially expedite the biofouling. This recycle system was used to check various controls such as no additives, cPANFs and heat-treated Mag-EPC together with Mag-EPC.

2.9. Two-vessel system for antifouling membrane.


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Figure 3B shows two-vessel system for the mechanistic study of enzymatic antifouling. MagEPC was confined in vessel #1 by using a magnet while the PVDF disc membranes attached on the slide glass was immersed into vessel #2. After the overnight culture of P. aeruginosa was inoculated, synthetic wastewater was continuously fed to the 1st vessel as an influent. The flow rate of both influent and effluent were 0.2 mL/min, and the working volume of each vessel was adjusted to 35 mL. The flow rate of internal recycle was also 0.2 mL/min, and the hydraulic residence time of each vessel was 175 min. At a specific time point during operation, the membrane was taken out and the surface biofouling was evaluated by measuring the change of permeability according to the procedure described in a previous study.28

2.10. Measurement of AHL autoinducers. The concentration of AHL autoinducers were measured by a colorimetric method as described previously.37-38 P. aeruginosa was cultured overnight in 29 mL of synthetic wastewater in 125 mL disposable flask (Corning, Corning, NY, USA) under shaking at 200 rpm. Synthetic wastewater was supplemented with 1 mL of test samples including bare cPANFs, heat treated Mag-EPC and Mag-EPC. When samples were applied, distilled water or PB for storage was replaced with synthetic wastewater. After centrifugation of overnight culture at 12,000 rpm for 30 min, cell pellet was discarded. This step was repeated twice. The supernatant was filtrated using 0.2 µm membrane (Merckmilipore, Billerica, MA, USA). 25 mL of supernatant and same volume of diethyl ether (Sigma Aldrich, St. Louis, MO, USA) were added to a sterilized tube. The tubes are kept on a rocking shaker at 50 rpm for 12 h. 17 mL of organic phase containing AHL autoinducers was transferred into a clean tube and evaporated via gentle heating using boiling water. 850 µL of dimethyl sulfoxide was added for re-solubilizing AHL autoinducers in a



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tube. 40 µL of the resolubilized solution was added into 96-well plate. Then, 50 µL of 1:1 mixture of hydroxyl amine (2M) and NaOH (3.5 M) was added. Subsequently, the same amount of 1:1 mixture of ferric chloride (10 % in 4 M HCl) and ethanol (95%) was mixed for color development. Absorbance was measured at 520 nm using a microplate reader (Infinite 200 PRO, TECAN, Hombrechtikon, Switzerland) for the quantification of AHL autoinducers.

3. RESULTS AND DISCUSSION

3.1. Acylase immobilization on cPANFs. Acylase was immobilized on cPANFs in the form of CA, EC, and Mag-EPC. The enzyme loading of each immobilization was estimated by measuring the amino acid contents after fully hydrolyzing the enzymes into amino acids.34 Usually, the enzyme loading of immobilized enzyme systems is indirectly estimated by measuring the enzyme amount in solution before/after immobilization and then calculating the disappeared enzyme amount from the solution upon enzyme immobilization. However, the enzyme loadings of EC and Mag-EPC are difficult to obtain due to the formation of insoluble crosslinked enzymes, which interfere with the correct measurement of enzyme amount via conventional protein assays based on soluble form of enzymes. As a bypass, we used the direct measurement of amino acid contents after the full hydrolysis of immobilized enzymes for the estimation of enzyme loadings. The estimated acylase loadings of CA, EC and Mag-EPC were 0.29, 0.46 and 2.2 mg acylase/mg cPANFs, respectively. The enzyme loading of Mag-EPC was 7.6 and 4.8 times higher than those of CA and EC, respectively, while EC resulted on 1.6-fold increase of enzyme loading when compared to CA. This 1.6-fold increase of enzyme loading via EC can be


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explained by the additional step of glutaraldehyde treatment, which crosslinks more enzyme molecules onto the covalently-attached enzyme molecules. On the other hand, the 4.8-fold increase of enzyme loading from EC to Mag-EPC can be explained by two factors. One is the enzyme precipitation upon the addition of ammonium sulfate, which allows for closer contact among precipitated enzyme molecules and efficient enzyme crosslinking as a result. The other factor would be the amine functionalized magnetic nanoparticles that can provide a lot more amino groups for crosslinking with enzyme molecules. Figure 4 shows the SEM images of cPANFs, CA, EC, and Mag-EPC, respectively. The average thickness of fibrous images was estimated by measuring the thickness of randomly selected 20 fibrous images using ImageJ software (National Institutes of Health, USA).39-40 The estimated average thicknesses of cPANFs, CA, EC, and Mag-EPC were 63.2 ± 10.6, 75.8 ± 16.2, 95.2 ± 21.9, and 169.3 ± 41.7 nm, respectively. An interesting observation is the increasing standard deviation from cPANFs to immobilized enzymes with higher enzyme loadings, which reflects the random conjugation of enzyme molecules by nature during the enzyme immobilization process. When compared to pristine cPANFs, Mag-EPC showed a significantly increased fiber thickness, and was also thicker than both CA and EC. This estimation of fiber thickness matches well with the enzyme loading results. The thick nanofibers of Mag-EPC imply that ammoniums sulfate precipitation and glutaraldehyde cross-linking improved the enzyme loading on cPANFs. Furthermore, the use of magnetic nanoparticles, allowing for facile magnetic separation (Figure S1), can further increase the enzyme loading on cPANFs due a lot more amino groups on MNPs that can form cross-linkages with enzyme molecules in a more efficient way.33



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3.2. Activity and stability of acylase-immobilizd cPANFs. The activities of CA, EC and Mag-EPC were determined via a fluorometric assay using the reaction of o-phthaldialdehyde with the amino group of L-methionine, which is generated from the acylase-catalyzed hydrolysis of N-acetyl-L-methionine. The measured activities of CA, EC and Mag-EPC samples were 1.2, 0.3 and 90 units per µg of cPANFs (Figure 5A). The activity of Mag-EPC per unit weight of cPANFs was 75 and 300 times higher than those of CA and EC, respectively. The specific activities of CA, EC and Mag-EPC, defined by the enzyme activity per unit weight of immobilized enzyme, were 4.1, 0.74 and 40 units/µg acylase, respectively. The specific activity of Mag-EPC was 9.8 and 54 times higher than those of CA and EC, respectively. This result suggests that the additional steps of enzyme precipitation and crosslinking for the preparation of Mag-EPC effectively protect the enzyme molecules from being denatured or inactivated during overall immobilization step. When considering the potential deformation of enzyme native structure upon chemical crosslinking, the enzyme precipitation step effectively prevents the enzyme inactivation by precipitating the enzyme molecules and allowing for efficient enzyme crosslinking. As a result, multiple chemical linkages on precipitated enzyme molecules would help to prevent the enzyme denaturation and inactivation. Interestingly, the specific activity of EC was 5.6 times lower than that of CA, revealing that the acylase molecules would be inactivated upon chemical crosslinking without being precipitated. We also obtained the kinetic parameters (kcat and KM) of free acylase and Mag-EPC. The kcat values of free acylase and Mag-EPC were 49.8 ± 2.2 and 37.4 ± 2.6 min-1, respectively. The reduced kcat of Mag-EPC can be explained by the less flexible acylase molecules of Mag-EPC upon enzyme crosslinking. The KM values of free acylase and Mag-EPC were 4.7 ± 1.3 and 5.8 ± 1.5 mM, respectively. The


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increased KM of Mag-EPC reveals the increased mass-transfer limitation due to the thick coating of crosslinked enzyme aggregates on the surface of cPANFs. Figure 5B shows the stabilities of free acylase, CA, EC and Mag-EPC in an aqueous solution at room temperature under rigorous shaking (200 rpm). After incubation for five days, the relative activities of free acylase, CA, EC and Mag-EPC were 9.3%, 8.8%, 29% and 65%, respectively. After 20 days, Mag-EPC maintained 55 % of initial activity, while CA and EC showed 3% and 16% of their initial activities, respectively. The higher stability of Mag-EPC than CA and EC is mainly due to the multi-point covalent linkages of enzyme molecules, which effectively prevent enzyme molecules from being structurally denatured and leached away. The step of enzyme precipitation by adding ammonium sulfate is critical for the efficient formation of chemical linkages during the follow-up enzyme crosslinking by shortening the distance among enzymes when enzyme molecules are precipitated.32-33 To investigate the contribution of enzyme leaching to the enzyme inactivation, the amount of leached enzymes was measured time-dependently and compared with the results of relative activity (Figure S2). Relative enzyme loading of each sample was calculated by the ratio of residual enzyme loading at each time point to the initial enzyme loading. In all the cases of CA, EC, and Mag-EPC, the enzyme leaching contributed to the decrease of residual enzyme activity, especially in the initial stage of incubation for the enzyme stability experiment. CA showed a vivid inactivation dominantly by the enzyme leaching from the first day of incubation. On the other hand, the initial inactivation of EC and Mag-EPC cannot be explained only by the enzyme leaching, which reveals another mechanism of enzyme inactivation via the inactivation of labile crosslinked enzymes upon enzyme crosslinking. Usually, chemical crosslinking of enzymes ends up with two different populations of crosslinked enzyme aggregates: one is a fairly stabilized



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form while the other is a labile form being inactivated during the initial stage of stability experiment. One of the most important criteria differentiating these two populations would be the number of chemical linkages on the surface of each enzyme molecules. In other words, the more number of chemical linkages on the enzyme surface would stabilize the enzyme activity more rigorously by preventing the enzyme denaturation more effectively. Interestingly, the stability of Mag-EPC shows bi-phasic inactivation curves with two different linear correlations in the semi-log scale plot (Figure 5B), which reveals two different populations of Mag-EPC. One is a labile form showing more rapid inactivation up to six days, and the halflife is calculated to be 8.4 days. The other population is fairly stable with a half-life of 27 days, covering the linear correlation in the semi-log scale plot after six days. This bi-phasic enzyme inactivation is frequently observed when chemical treatment such as enzyme crosslinking is performed.33 Upon enzyme crosslinking, higher degree of crosslinking forming multi-point chemical linkages on the enzyme surface would generate a highly-stabilized population, while enzymes with smaller number of chemical linkages or deformation would inactivate during the initial stage of incubation. The half-lives of CA and EC, obtained from the linear correlation during the initial stage of incubation, were 0.57 days and 2.9 days, respectively. When compared to Mag-EPC with multiple chemical linkages upon enzyme crosslinking, the CA approach provides only one or limited number of chemical linkages that cannot provide good stabilization mechanism. On the other hand, EC shows a marginal stabilization than CA by allowing multiple chemical linkages, but is less stable than Mag-EPC by not having the enzyme precipitation that can improve the chance of more chemical linkages on each enzyme molecule. We further checked the enzyme stability in the synthetic wastewater26 with high level of organic pollutants. After incubation for five days, CA, EC, and Mag-EPC maintained 3, 25 and


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30% of their initial activities, respectively (Figure S3). This implies that enzyme crosslinking has a critical role for the enzyme stabilization even in harsh conditions, such as wastewater containing various organic pollutants.

3.3. Biofilm control by Mag-EPC under static condition. The effect of Mag-EPC on biofilm formation was evaluated using microplate assay under static condition (Figure 6A).35-36 Mag-EPC was added to the microplate well at final concentration of 0, 5, 10, 20 and 40 µg/mL, and the biofilm formation in the internal surface of each well was checked by measuring the absorbance at 590 nm after the crystal violet staining. As a result, the higher concentration of Mag-EPC has showed a more inhibitory effect on the biofilm formation (Figure 6B). For example, the well with 40 µg/mL of Mag-EPC showed 5 times lower absorbance at 590 nm than the well without Mag-EPC, which represents more effective inhibition of biofilm formation in the presence of Mag-EPC. On the other hand, cPANFs with no immobilized acylase did not show any inhibition of biofilm formation (Figure 6B). In other words, immobilized and stabilized acylase in the form of Mag-EPC effectively inhibits

the

biofilm

formation

potentially

via

their

catalytic

hydrolysis

of

AHL

autoinducers.41,28,42

3.4. Antifouling of Mag-EPC under continuous membrane filtration. The antifouling of Mag-EPC was investigated using the system of continuous membrane filtration, where the membrane biofouling can be quantitatively estimated by the increase of trans-membrane pressure (TMP) (Figure 3A). Figure 7A clearly shows that Mag-EPC delayed the increase of TMP more effectively as the concentration of added Mag-EPC was increased, which is a similar trend to the effect of Mag-EPC against the biofilm formation under static



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incubation (Figure 6B). In other words, the delayed increase of TMP in the presence of MagEPC can be explained by the quorum quenching of Mag-EPC that can inactivate the AHL autoinducers.28,41-42 We also found that the recycle of filtrate vividly expedited the membrane fouling even in the presence of Mag-EPC (Figure S4). This result can be explained by the recycle of autoinducers in the filtrate that can speed up the membrane fouling. Under the recycle of filtrate, the continuous membrane filtration was performed to check several cases of controls, such as no additives, cPANFs, and heat-treated Mag-EPC (Figure 7B). Both cPANFs and heat-treated Mag-EPC marginally delayed the increase of TMP, but their effects are so minimal when compared to the case of Mag-EPC addition. Marginal delay of TMP increase in the presence of cPANFs can be partly explained by the physical effects of cPANFs on the surface of membrane, which can delay the membrane fouling as well as the biofilm formation. However, the vivid difference between mag-EPC and heat-treated Mag-EPC in delaying the TMP increase clearly reveals that the membrane fouling is delayed by the catalytic action of acylase in the form of Mag-EPC, hydrolyzing the AHL autoinducers and quenching the quorum sensing.

3.5. Mechanisms of antifouling in the presence of Mag-EPC. For the investigation of mechanism for the antifouling of Mag-EPC, we developed a twovessel system (Figure 3B) where Mag-EPC and membranes are separated into two different vessels. Mag-EPC can be confined in the first vessel by using a magnet while the second vessel can contain multiple membranes by attaching them on slides. By this way, we can easily increase the number of membranes to be tested for the mechanism study of antifouling in the presence of Mag-EPC. The control operation with no addition of Mag-EPC showed 31% of initial permeability from the first data point of one day, representing a drastic permeability loss. On the 18 Environment ACS Paragon Plus

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other hand, the addition of Mag-EPC in the first vessel maintained 75% of initial membrane permeability even after operation for four days (Figure 8). Considering that membranes were just immersed in the working fluid containing P. aeruginosa, the reduced permeability was mainly caused by the biofilm growth on the surface of membrane. Furthermore, the cell density in each vessel was maintained to the same level due to the internal recycling between the vessel #1 & 2 with same working volume. The only difference is the presence of Mag-EPC, which is known to degrade the AHL autoinducers and quench the quorum sensing.28,41-42 Therefore, AHLs were continuously degraded by Mag-EPC in the vessel #1, and this catalytic action led to relatively low AHL concentration in the overall bulk phase, including the vessel #2. Although Mag-EPC was retained in vessel #1 by a magnet without sufficient mixing, it seems that high enzyme loading on cPANFs can enable the hydrolysis of AHL autoinducers and suppress the biofilm formation. As a result, it was experimentally demonstrated that the quorum quenching effect of Mag-EPC effectively controls the membrane surface biofouling even in a separated vessel, which has opened up a convenient option of enzymatic quorum quenching in the real engineering system of waste treatment. To check the degradation of AHL autoinducers in the presence of Mag-EPC, P. aeruginosa was incubated in synthetic wastewater under four different cases: 1) no additives, 2) cPANFs, 3) heat-treated Mag-EPC, and 4) Mag-EPC. The amount of AHLs was quantified by the combined protocol of liquid/liquid extraction and colorimetric assay.37-38 When P. aeruginosa was incubated in synthetic wastewater with cPANFs, heat treated Mag-EPC and Mag-EPC, the amount AHL autoinducers was reduced by 7%, 27% and 78%, when compared to no additives, respectively (Figure S5). These results confirm that Mag-EPC can reduce the amount of AHL autoinducers based on the catalytic hydrolysis of AHL autoinducers.



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4. CONCLUSIONS We have developed the highly effective antifouling platform by immobilizing and stabilizing a quorum quenching enzyme. Acylase, as one of quorum quenching enzymes, was immobilized on carboxylated polyaniline nanofibers (cPANFs) via enzyme precipitate coating method (MagEPC). Mag-EPC can be easily captured by a simple magnet capture, and improved both loading and stability of enzymes. The antifouling capacity of Mag-EPC was tested in various systems using a model microorganism of P. aeruginosa. Mag-EPC effectively alleviated the biofilm formation on the inner surface of microplate wells, and demonstrated antifouling capacity against the membrane biofouling in the home-built single-vessel and two-vessel systems. The unique antifouling feature of Mag-EPC, originated from its catalytic hydrolysis of AHL antoinducers, has a great potential to be used for the treatment of wastewaters. It is also anticipated that nanobiocatalytic approach of Mag-EPC on cPANFs can be employed for the immobilization and stabilization of enzymes for various enzyme applications, where the poor enzyme stability as well as the low enzyme loading has hampered the practical uses of enzymes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Magnetic separation of Mag-EPC, Comparison of relative enzyme activity and loading, Stability in the synthetic wastewater, Trans-membrane pressure under filtrate


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discharge and recycling conditions, AHL generation after overnight culture of P. aeruginosa.

AUTHOR INFORMATION Corresponding authors *Jungbae Kim. E-mail: [email protected] *Kyung-Min Yeon. E-mail: [email protected] Notes The authors declare no competing financial interest

ACKNOWLEDGEMENTS This research was supported by Global Research Laboratory Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2014K1A1A2043032).



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Figure Legends

Figure 1. Schematic illustrations for enzymatic antifouling of Mag-EPC

Figure 2. Schematic illustration for the immobilization of acylase on cPANFs via three different methods of covalent attachment (CA), enzyme coating (EC) and magnetically separable enzyme precipitate coating (Mag-EPC).

Figure 3. Schematic illustrations for (A) one vessel system of membrane filtration and (B) two vessel system where antifouling Mag-EPC (vessel #1) and membrane (vessel #2) were separated.

Figure 4. SEM images of (A) cPANFs, (B) CA, (C) EC, and (D) Mag-EPC.

Figure 5. (A) Activities of CA, EC and Mag-EPC, and (B) stabilities of free acylase, CA, EC and Mag-EPC. One unit will hydrolyze 1.0 µmole of N-acetyl-L-methionine per hour at pH 7.0 at 25°C.

Figure 6. (A) Schematic illustration for the evaluation of biofilm growth in a microplate under static condition. (B) Biofilm quantification at different dosing concentrations of bare cPANFs and Mag-EPC.

Figure 7. (A) Trans-membrane pressure (TMP) at different concentrations of Mag-EPC. (B) TMP in the presence of Mag-EPC and three controls under continuous membrane filtration.



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Figure 8. Biofilm formation on membranes, represented by the decrease of permeability, when Mag-EPC and membranes are separated in two different vessels. Relative permeability is the ratio of membrane permeability at a specific time to that of initial start.


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Figure 1. Schematic illustration for enzymatic antifouling of Mag-EPC.



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Figure 2. Schematic illustration for the immobilization of acylase on cPANFs via three different methods of covalent attachment (CA), enzyme coating (EC) and magnetically separable enzyme precipitate coating (Mag-EPC).


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Figure 3. Schematic illustrations for (A) one vessel system of membrane filtration and (B) two vessel system where antifouling Mag-EPC (vessel #1) and membrane (vessel #2) were separated.



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Figure 4. SEM images of (A) cPANFs, (B) CA, (C) EC, and (D) Mag-EPC.


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Figure 5. (A) Activities of CA, EC and Mag-EPC, and (B) stabilities of free acylase, CA, EC and Mag-EPC. One unit will hydrolyze 1.0 µmole of N-acetyl-L-methionine per hour at pH 7.0 at 25°C



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Figure 6. (A) Schematic illustration for the evaluation of biofilm growth in a microplate under static condition. (B) Biofilm quantification at different dosing concentrations of bare cPANFs and Mag-EPC.


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Figure 7. (A) Trans-membrane pressure (TMP) at different concentrations of Mag-EPC. (B) TMP in the presence of Mag-EPC and three controls under continuous membrane filtration.



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Figure 8. Biofilm formation on membranes, represented by the decrease of permeability, when Mag-EPC and membranes are separated in two different vessels. Relative permeability is the ratio of membrane permeability at a specific time to that of initial start.


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Table of Contents


Immobilization and Stabilization of Acylase on Carboxylated

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