Article pubs.acs.org/Biomac
Immobilization to Positively Charged Cellulose Nanocrystals Enhances the Antibacterial Activity and Stability of Hen Egg White and T4 Lysozyme Adel Abouhmad,†,‡ Tarek Dishisha,§ Magdy A. Amin,∥ and Rajni Hatti-Kaul*,†
Biomacromolecules 2017.18:1600-1608. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/06/19. For personal use only.
†
Biotechnology, Department of Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden ‡ Department of Microbiology and Immunology, Faculty of Pharmacy, Al-Azhar University, 715 24 Assiut, Egypt § Department of Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef University, 625 11 Beni-Suef, Egypt ∥ Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, 115 62 Cairo, Egypt S Supporting Information *
ABSTRACT: Antibacterial bionanostructures were produced from cellulose nanocrystals (CNC) with immobilized lysozyme from hen egg white (HEW) and T4 bacteriophage, respectively. The nanocrystals were prepared from microcrystalline cellulose by ammonium persulfate oxidation with a yield of 68% and having an average size of 250 nm and low polydispersity index. HEW lysozyme (HEWL) and T4 lysozyme (T4L) were immobilized to CNC by different mechanisms including adsorption and covalent coupling to carbodiimide-activated carboxylate groups and to glutaraldehyde-activated aminated CNC (Am-CNC), respectively. The effect of immobilization on the enzymatic activity (both lytic and hydrolytic) and antibacterial activity of the lysozymes was studied using different methods. Am-CNC-lysozyme conjugates retained the highest lytic activity, 86.3% and 78.3% for HEWL and T4L, respectively. They also showed enhanced bactericidal activity with high potency against Grampositive as well as Gram-negative bacteria in a relatively shorter time as compared to the free enzymes and resulted in extensive cellular damage, as shown by transmission electron microscopy. The enhanced antibacterial activity was correlated with the increase in zeta potential of Am-CNC-lysozyme conjugates. The immobilized lysozyme preparations further exhibited enhanced storage stability at 4 and 22 °C.
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INTRODUCTION
general, nanoparticles mediate their effect by disruption of the membrane potential and integrity or generation of reactive oxygen species leading to oxidative stress and damage to cell components. 2,4 They are further used as carriers of antimicrobial peptides and enzymes.5 Since their size is proportionate to that of the bacterial cells and their surface
The concern over the rising incidence of antibacterial resistance and emergence of “superbugs” as a result of rampant use of antibiotics has resulted in an urgent demand for alternative therapies.1 Several strategies are being pursued to search for and develop novel, effective antimicrobials such as nanoparticles and antimicrobial peptides and enzymes. Antimicrobial nanoparticles are based on metals, metal oxides, and organic materials, and their activity is determined by their intrinsic and modified physicochemical properties.2,3 In © 2017 American Chemical Society
Received: February 11, 2017 Revised: March 28, 2017 Published: March 28, 2017 1600
DOI: 10.1021/acs.biomac.7b00219 Biomacromolecules 2017, 18, 1600−1608
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can be tailored, nanoparticles can be a potential platform for extra- and intracellular delivery of the antimicrobials with enhanced efficacy and low risk of resistance.6,7 A limitation in the use of nanoparticles, especially the inorganic particles, but even certain organic materials could, however, be their potential toxicity and nonselective damage to mammalian cells.3,8,9 This has rekindled interest in the fabrication of safe, biocompatible, and biodegradable nanoparticles.10 Cellulose nanocrystals (CNC) represent a new biological class of nanomaterials with versatile formulations as nanocomposites, nanofillers, adsorbents, gene carriers and polymer reinforcement materials.11 They have attracted interest in recent years for a variety of applications; in biomedical engineering, biosensing, catalysis, material science, electronics, adsorption of dyes, and so on.12 The attractive features of CNC are their renewable origin, low cost, ease of preparation and modification, biocompatibility, biodegradability, and high mechanical and chemical strength, among others.13 Moreover, the high stability of CNC against proteolytic enzymes, acids, and temperature make them attractive as carriers for immobilization of enzymes as well as for targeted drug delivery.14−17 Toxicity assessments conducted, for example, on brain microvascular endothelial cells have indicated the CNC to be safe without any health or environmental impact.18 Lysozymes, also known as muramidases, are well-known antibacterial enzymes that hydrolyze β-1,4-glycosidic bonds between N-acetylglucosamine (NAG) and N-acetyl muramic acid (NAM) in the peptidoglycan layer of the cell wall, especially that of Gram-positive bacteria, resulting in cell lysis and bacterial death.19 Hen egg white is a rich source of lysozyme; the enzyme is used widely in food and pharmaceutical applications.20 T4 bacteriophage, that infects Escherichia coli bacteria, produces lysozyme to facilitate the release of the virion progeny from the infected cells.21 It attacks Gram-positive bacteria through its muramidase activity22 and Gram-negative bacteria through its membrane disruption ability.23 Hen egg white and T4 lysozyme differ in their primary structures but have similarities in their overall backbone conformation, mode of substrate binding, and also the catalytic action.24 Hen egg white lysozyme has been immobilized by different techniques to a variety of matrices, including different nanosupports such as carbon nanotubes,25 chitosan nanofibers,26 gold nanowires,27 nano diamonds,28 magnetic Fe3O4,29 ZnO,30 silver,31 silica,32,33 and polymeric nanoparticles.6 On the other hand, immobilization of T4 lysozyme has been limited to a few studies, aiming at studying the impact on protein conformation, activity, and stability.34−36 Recently, we reported affinity immobilization of T4 lysozyme to cellulosic wounddressing material through the fusion tag of cellulose binding domain at the C-terminal of the enzyme; the enzyme was strongly adsorbed, with retention of 99.3% of its bactericidal activity against Gram-positive bacteria.37 In the present report, we describe a study on immobilization of hen egg white and T4 lysozymes to cellulose nanocrystals through different mechanisms and demonstrate that the surface modification and the mode of immobilization are critical for the retention of the enzymatic (lytic and hydrolytic) and antibacterial activity as well as stability of the immobilized enzymes.
Article
EXPERIMENTAL SECTION
Materials. Hen egg white lysozyme (HEWL), microcrystalline cellulose powder (cotton linters), bicinchoninic acid (BCA) reagent, glycol chitin (GC), (N-morpholino) ethanesulfonic acid (MES), Nhydroxysulfosuccinimide (NHS), 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC), and lyophilized Micrococcus lysodeikticus cells were obtained from Sigma-Aldrich (St Louis, MO, U.S.A.). Ammonium persulfate (APS) was purchased from Merck (Darmstadt, Germany). Cloning, Expression, and Production of T4L. The gene encoding T4L was cloned, expressed, and the recombinant enzyme was purified as reported earlier.37 The purified enzyme was freezedried (Labconco Freeze-Dryer, FreeZone 4.5, Labconco, MO, U.S.A.), and stored at −20 °C. Directly before use, the enzyme was dissolved in 10 mM sodium phosphate buffer, pH 7.4, to the desired protein concentration and kept at 4 °C. Preparation of CNC. CNC were prepared as reported earlier;38 10 g microcrystalline cellulose powder was suspended in 1 L of 1 M APS solution (pH 2) and heated to 60 °C for 16 h with stirring. The suspension was centrifuged (15000 g, 10 min, 4 °C; Sorvall Lynx 4000 centrifuge, Thermo Scientific, Waltham, MA, U.S.A.), the supernatant was decanted, and the pellet of nanocrystals was washed repeatedly with deionized water with intermittent centrifugation until the solution pH was ∼4. The pellet was freeze-dried and stored at −20 °C. Prior to use, CNC were suspended in 10 mM sodium phosphate buffer pH 7.4, and sonicated on ice (3 × 3 min, cycles 0.7) with UP400S sonicator (Dr. Hielscher GmbH, Stahnsdorf, Germany) to obtain fine and homogeneous dispersion. To obtain CNC as ammonium salt (CNCCOO-NH4), the suspension was neutralized with NH4OH (110 mL) until pH 8, followed by centrifugation/washing cycles with deionized water.39 Preparation of Amino-Functionalized CNC. Amino-functionalized CNC (Am-CNC) were prepared by first oxidizing CNC-COONH4 to dialdehyde-CNC (Diald-CNC), followed by amination.40 For this, CNC-COO-NH4 was washed extensively with water, then 250 mL of the suspension (0.4 wt %) was mixed with 0.16 M sodium metaperiodate, and stirred at room temperature in dark for 48 h. Excess sodium meta-periodate was removed by addition of 15 mL ethylene glycol. Diald-CNC were recovered by centrifugation (15000 g, 10 min, 4 °C), dialyzed against water using 12000−14000 molecular weight cut off dialysis membrane (Spectra/Pro 4, Spectrum Laboratories, Rancho Dominguez, CA, U.S.A.) for 3 days, and then stored at 4 °C. Amination of Diald-CNC was done directly before use, by mixing 100 mL of the suspension (0.5 wt %) with 20 mM ethylenediamine solution pH 10 with stirring at room temperature for 8 h. The mixture was subjected to in situ reduction by addition of 0.58 g sodium borohydride with stirring for 4 extra hours, followed by repeated washing as above until the solution reached neutral pH. Am-CNC preparation was stored at 4 °C until the subsequent step of enzyme immobilization. Characterization of CNC. The morphology of CNC was characterized using transmission electron microscopy (TEM). A total of 2 μL of CNC dispersion (0.1 wt % in water) was mounted on pioliform-coated TEM copper grid, set to dry, and examined with TEM (JEOL, JEM-1230 Jeol, Tokyo, Japan) run at 80 kV. Areas of interest were photographed and recorded with a Gatan MultiScan 794 CCD camera. Dynamic light scattering (DLS) Malvern Zetasizer Nano-S (Malvern Instruments, Malvern, U.K.) was applied for determination of hydrodynamic size distribution of CNC. A total of 1 mL of sonicated CNC dispersion (0.1 mg/mL) was placed in a disposable plastic cuvette, data was collected after 2 min equilibration time and averaged over 12 measurements at a backscatter angle of 173°. The particle size distribution was characterized by intensity-averaged mean and polydispersity index. The surface charge of pristine CNC and CNC with immobilized lysozymes was estimated by measuring the zeta potential ζ (Zetasizer Nano ZS, Malvern Instruments) in disposable zeta cells with 1 mL of 0.1 mg/mL CNC dispersion diluted in 10 mM sodium phosphate 1601
DOI: 10.1021/acs.biomac.7b00219 Biomacromolecules 2017, 18, 1600−1608
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Biomacromolecules buffer, pH 7.4, at 25 °C. The data was reported as the average and standard deviation of measurements of three freshly prepared samples. Immobilization of T4L and HEWL. Different immobilization techniques were employed for immobilization of T4L and HEWL to CNC preparations. Prior to immobilization, 50 mg CNC preparation was washed with water followed by 10 mM sodium phosphate buffer, pH 7.4. The enzymes were also dissolved in the same buffer. Physical Adsorption to CNC.28 A total of 50 mg CNC was incubated overnight with 3 mL of HEWL (4 mg/mL) and T4L (2 mg/ mL), respectively, at 4 °C on a rocking table. CNC with bound HEWL and T4L were collected by centrifugation (3900 g, 4 °C for 20 min, Sigma 3−16PK), followed by 3 cycles of repeated washing (20 min each) and centrifugation to remove any unbound proteins, and then stored at 4 °C for further characterization. To study the effect of pH on the adsorption of the enzymes, the immobilization was carried out at pH range of 4−10 using 10 mM sodium acetate buffer, pH 4−5, 10 mM sodium phosphate buffer, pH 6−7.4, and 10 mM Tris-HCl buffer, pH 9−10, respectively. Covalent Immobilization. Covalent coupling of the lysozymes to the nanocrystals was performed using two different methods. The first method involved activation of the carboxyl groups of CNC with carbodiimide (EDC), followed by incubation with the protein solutions where the coupling reaction occurs with the protein amino groups.42 A total of 1 g CNC was suspended in 100 mL of 0.05 M MES/0.05 M NaCl buffer, pH 5.7, prior to addition of EDC and 10 mM NHS and stirred at room temperature for 3 h. The activated preparation was collected by centrifugation (15000 g, 10 min, 4 °C), washed repeatedly with deionized water until the pH of the solution reached ∼6, and then lyophilized to yield a white powder. Subsequently, this powder was suspended in 10 mM sodium phosphate buffer, pH 7.4, and used for protein conjugation overnight at 4 °C, followed by centrifugation and washing as described above. The second method involved coupling of the lysozymes to glutaraldehyde-activated Am-CNC. A total of 200 mg Am-CNC suspended in 10 mL of 10 mM sodium phosphate buffer, pH 7.4, were mixed with 750 μL of glutaraldehyde, and stirred at room temperature for 1 h. Excess glutaraldehyde was removed by washing several times with the same buffer. The nanocrystals were then incubated overnight with the lysozymes in the phosphate buffer, pH 7.4, at 4 °C, centrifuged, and washed as described above, and then stored at 4 °C as described above. Determination of Immobilization Yield and Binding Capacity. The immobilization yield was determined by calculating the difference between the initial amount of the protein in solution before immobilization and the residual protein in the supernatant and washings after immobilization. The protein content was determined by BCA method according to the manufacturerś instructions.
the turbidimetric (using lyophilized M. lysodeikticus cells and chloroform treated E. coli B cells as substrate for HEWL and T4L, respectively) and lysoplate (using autoclaved M. lysodeikticus cells) assays and the hydrolytic activity against glycol chitin. The details of the assays are provided in Supporting Information. All assays were performed in triplicates. The turbidimetric assays were used to determine the effect of pH, temperature, and storage stability on the enzymatic activity of the free and immobilized lysozymes. For studying the effect of pH, the assays were performed at 25 °C in a pH range 4−10 using 10 mM of sodium acetate buffer pH 4−5, sodium phosphate buffer pH 6−7.4, or TrisHCl buffer pH 9−10. The effect of temperature was tested through incubation of free HEWL (1 μg) and T4L (3 μg), and CNC-lysozyme preparations (1 mg) in a temperature range of 25−90 °C for 5 min, followed by cooling the reaction mixture and measuring the residual activity under standard assay conditions. Stability of the different lysozyme preparations was tested during storage in 10 mM sodium phosphate buffer, pH 7.4, at room temperature and at 4 °C, respectively, for 60 days. Samples were withdrawn periodically for determination of residual activities. Residual enzymatic activity was calculated as a percentage of the initial activity of the preparations. Activity results are an average of triplicate measurements of independent replicates. residual activity% activity of the CNC − lysozyme conjugates after storage = × 100 initial activity of the CNC − lysozyme conjugates
Evaluation of the Antibacterial Activity of Free and Immobilized Lysozymes. The antibacterial activities of free HEWL and T4L, as well as the enzymes immobilized by adsorption to CNC and covalent coupling to Am-CNC, respectively, were determined by microdilution checkerboard assay, time-kill assay with ALAMARBLUE, cell viability analysis using bacterial viability stain kit, and TEM examination (see Supporting Information for detailed description of methods). Gram-negative bacteria E. coli and Pseudomonas mendocina (ATCC 25413), and Gram-positive bacteria M. lysodeikticus (ATCC 4698) and Corynebacterium sp. (ATCC 21245) were used as test strains.
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RESULTS AND DISCUSSION Preparation and Characterization of CNC and CNCLysozyme Conjugates. CNC was prepared from microcrystalline cellulose through facile single step APS oxidation procedure. The method has significant benefits in terms of low cost, scalability, mild reaction conditions and high yield38,39,41,42 as compared to the other methods, including acid hydrolysis,43−45 oxidation,46 or high intensity ultrasonication.47 Moreover, the method yields nanocrystals with exposed carboxyl groups, which can be reacted to achieve surface modification and protein immobilization.48 CNC were obtained with a yield of 68%, which is higher than that reported earlier (yields ranging between 22 and 61%).38,41,49 TEM showed the CNC to be rod shaped with a size range of 200−300 nm (Figure S1, Supporting Information). The narrow hydrodynamic size distribution was further confirmed by DLS; the mean CNC size was ∼250 nm with ∼85% intensity and polydispersity index of 0.1 (Figure S2, Supporting Information). The nanocrystals form a stable aqueous suspension due to the repulsive forces that prevent aggregation of particles,43,40 and further neutralization with NH4OH improved the dispersion in accordance with an earlier report.39 We chose to investigate different modes of immobilization of HEWL and T4L to cellulose nanocrystals and their impact on enzymatic and antibacterial activity of the immobilized enzymes. Adsorption of the enzymes was primarily based on electrostatic interactions between the negatively charged
immobilization yield(%) amt of protein before binding − amt of residual protein after binding = amt of protein before binding × 100
The binding capacities of the CNC using different immobilization techniques were determined through overnight incubation of 10 mg of the nanocrystals with increasing concentrations of lysozymes (in 10 mM sodium phosphate buffer, pH 7.4) at 4 °C with shaking. Subsequently, the CNC were washed and the amount of immobilized protein was determined as described above. The binding capacity was calculated as follows:
binding capacity(mg protein/mg CNC) =
amt of immobilized protein(mg) wt of CNC(mg)
For each experiment, the measurements were performed in triplicates with corresponding blank. Determination of the Enzymatic Activity of Free and Immobilized Lysozymes. The catalytic activity of the enzyme preparations was measured as the lytic activity against bacterial cells by 1602
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Biomacromolecules Table 1. Immobilization of Hen Egg White and T4 Lysozymes by Different Mechanisms to Cellulose Nanocrystalsa immobilization methodb
immobilization yieldb (%)
total protein immobilized (mg)
activityc (U/mg CNC)
specific activityd
retained activitye (%)
211.2 112.8
10.6 5.6
46.3 × 103 38.0 × 103
9.8 × 103 4.3 × 103
24.5 0.5 × 10−2
240.0 120.0
12.0 6.0
25.0 × 103 0.0
6.0 × 103 0.0
15 0.0
230.4 110.4
11.5 5.5
149.8 × 103 58.8 × 108
34.5 × 103 6.5 × 108
86.3 78.3
amt protein immobilized (mg/g CNC)
Physical Adsorption to CNC at pH 7.4 HEWL 88 T4L 94 Covalent Coupling to EDC-Activated CNC HEWL 100 T4L 100 Covalent Coupling to Am-CNC HEWL 96 T4L 92 a
Results in the table are the average of triplicates. bHEWL (12 mg) and T4L (6 mg) were mixed with 50 mg CNC. cEnzymtic activity was determined through turbidimetric assay against lyophilized M. lysodeikticus and chloroform-treated E. coli B cells, as substrates for HEWL and T4L, respectively. The activity of the immobilized enzyme was obtained from measuring the activity of unbound enzyme. dActivity units per mg immobilized enzyme. Specific activities of free HEWL and T4L were 40 × 103 and 8.3 × 108 U per mg enzyme. eActivity of enzyme after binding/ activity of the enzyme before binding with turbidimetric assay under the same conditions.
nanocrystals and the enzymes with a net positive charge (isoelectric point = ∼11.16 for HEWL and ∼9.59 for T4L). The other two covalent coupling methods were based on different chemistries. One involved EDC-activation of the carboxyl groups on CNC to form the O-isoacylurea intermediate that reacts with the amine groups to form amides. In the other method, the sugar residues in the CNC were oxidized by meta-periodate to yield dialdehyde groups that were further reacted to generate amino functionalities and thereafter activated with glutaraldehyde for reaction with amino groups on the enzymes. The immobilization was measured in terms of the amount of bound protein and retained activity (using the turbidimetric method) with respect to the free enzymes using M. lysodeikticus and chloroform-treated E. coli B cells as substrates for HEWL and T4L, respectively. Adsorption of the enzymes was investigated at different pH values ranging from 4 to 10. HEWL showed highest immobilization yield at pH 9 (228 mg/g CNC), but the highest retained activity was 24.5% of the initial enzyme load at pH 7.4. In case of T4L, the highest immobilization yield was obtained at pH 7.4 (113 mg/g CNC) but with negligible activity at all pH values (Table S1, Supporting Information). Covalent coupling of the lysozymes to the EDC-activated nanocrystals resulted in quantitative yield of the immobilized proteins (240 mg/g for HEWL and 120 mg/g for T4L) but with only 15% of the activity of free HEWL and no activity for T4L. On the other hand, covalent coupling to glutaraldehyde activated Am-CNC gave high immobilization yields of 96% for HEWL (230 mg/g) and 92% for T4L (110 mg/g), and their corresponding retained activity were 86.3% and 78.3% (Table 1). Comparison of the binding capacity for HEWL with that on other nanostructures such as diamond nanocrystals,28 electrospun chitosan nanofibers,50 ZnO,51 TiO2,52 polystyrene,6 and silica53 showed that significantly higher amounts of the protein could be loaded on the CNC preparations in this study. The immobilized lysozyme preparations obtained by physical adsorption and covalent coupling to Am-CNC were further characterized with respect to certain parameters and compared with the free enzymes. Table 2 shows the zeta potential (ζ) values of the original and modified nanocrystals and of free and immobilized enzymes. The highly negatively charged CNC formed with exposed carboxylate groups was confirmed by the ζ value of −54 mV,54 and on treating the particles with NH4OH, the value was increased to −42 mV. On the other hand, amino-functionalization of the CNC resulted in a
Table 2. Zeta Potential (ζ) in Millivolt (mV) of CNC and CNC with Immobilized Lysozymes samples
zeta potential (mV)
CNC CNC-COONH4+ Am-CNC HEWL T4L CNC with adsorbed HEWL CNC with adsorbed T4L Am-CNC with bound HEWL Am-CNC with bound T4L M. lysodeikticus cells chloroform-treated E. coli B cells
−54.0 ± 3.0 −42.0 ± 0.9 +31.0 ± 1.2 +9.5 ± 1.6 +6.8 ± 1.4 −31.0 ± 1.3 −32.4 ± 4.0 +37.2 ± 1.1 +42.6 ± 2.4 −26.0 ± 2.2 −21.0 ± 0.8
dramatic transformation of the charge of the particles (ζ potential of +31 mV). Immobilization of HEWL and T4L (with individual ζ of +9.5 and +6.8 mV, respectively) to the different nanocrystals resulted in further increase in ζ to +37.2 mV for Am-CNC-HEWL and +42.6 mV for Am-CNC-T4L (Table 2). The initial electrostatic interaction between lysozymes and the negatively charged cell wall is considered to be the main driving force controlling the bacterial lysis, which would explain the high lytic activity of the Am-CNC-lysozymes with highly positive ζ value (Tables 1 and 2). This is in accordance with the previous reports in which HEWL immobilized to positively charged polystyrene latex and ZnO nanoparticles, respectively, showed enhanced lytic activity.6,26 Also, the poor activity of the physically adsorbed enzymes could be attributed to the loss of their positive charge, evidenced by the decrease in the ζ values on immobilization from +9.5 to −31.0 mV in case of HEWL and from +6.8 to −32.4 mV for T4L (Table 2). The pH profiles of the immobilized lysozymes showed a shift in the optimum pH of the enzymatic activity toward the alkaline side with respect to that of the free enzymes, from pH 6.5 to 7.4 for HEWL and from pH 7.4 to 8.5 for T4L (Figure S3, Supporting Information). This shift in the apparent pH optimum is likely to be due to involvement of amino groups on the enzymes in binding in both cases, resulting in a relative increase in the number of acidic groups and thus the negative charge on the enzyme surface. To study the effect of immobilization on thermal stability, the enzyme preparations were exposed to different temperatures for 5 min prior to measuring the residual enzymatic 1603
DOI: 10.1021/acs.biomac.7b00219 Biomacromolecules 2017, 18, 1600−1608
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Figure 1. Storage stability of (a) HEWL at 4 °C, (b) HEWL at 25 °C, (c) T4L at 4 °C, and (d) T4L at 25 °C, in 10 mM sodium phosphate buffer, pH 7.4. The samples were taken periodically and residual enzymatic activity was determined at 25 °C by turbidimetric assay on lyophilized M. lysodeikticus and E. coli B cells, respectively, as substrates and expressed as a percentage of the initial enzymatic activity. The experiment was performed in triplicates, with triplicate measurements for each data point.
activity. The data revealed that the thermal stability of HEWL was enhanced upon immobilization; free HEWL lost its activity completely at 70 °C, while the adsorbed and covalently coupled enzyme preparations retained 20% and 55% activity, respectively (Figure S4, Supporting Information). T4L was more sensitive to increase in temperature as compared to HEWL, and no notable change in its stability was observed upon immobilization (Figure S4, Supporting Information). The immobilized lysozyme preparations exhibited higher stability than the free counterparts when stored at 4 and 22 °C. While soluble HEWL lost its enzymatic activity after 20 days at 22 °C and after 35 days at 4 °C, immobilized HEWL by adsorption and coupling to Am-CNC retained 40% and 50% of their original activity at 22 °C, and 60% and 75%, respectively, at 4 °C after 60 days (Figure 1a,b). Soluble T4L was more unstable than HEWL and lost its enzymatic activity completely within 2 weeks at 22 °C and after 25 days at 4 °C, but the residual activity of the adsorbed and covalently bound T4L was higher than immobilized HEWL, displaying 50% and 60% activity, respectively, at 22 °C, and 80% and 70% activity, respectively, at 4 °C, after 60 days (Figure 1c,d). Hydrolytic Activity of the Immobilized Lysozymes. The enzymatic activity of the immobilized lysozyme preparations was also determined by the lysoplate assay using the autoclaved M. lysodeikticus cells as the substrate and by hydrolysis of the soluble substrate, glycol chitin. As seen in Table 3, the lysoplate assay in all cases showed remarkably higher activity than that obtained with the whole cells by the turbidimetric assay (data not shown). Interestingly, even the T4 lysozyme bound to EDC-activated nanocrystals that showed no activity against the chloroform treated E. coli cells, exhibited 86% activity retention by the lysoplate assay. This is because the peptidoglycan layer of the M. lysodeikticus cells in the
Table 3. Retained Activities of Free and Immobilized HEW and T4 Lysozymes Measured by Different Assays % retained activity determined by enzyme preparation HEWL free enzyme adsorbed (pH 7.4) coupled to EDC activated CNC Coupled to Am-CNC T4L free enzyme adsorbed (pH 7.4) coupled to EDC-activated CNC coupled to Am-CNC
turbidimetric assaya
lysoplate assay
glycol chitin
100.0 24.5 15.0 ± 3.1
100.0 88.8 ± 1.3 90.3 ± 1.7
100.0 92.0 ± 2.2 95.0 ± 0.7
86.3 ± 1.5
97.4 ± 2.1
98.0 ± 0.4
100.0 0.5 × 10−2 0.0
100.0 60.0 ± 1.6 86.0 ± 0.4
100.0 72 ± 2.7 89 ± 0.5
78.3 ± 2.8
98.0 ± 1.8
95 ± 2.1
a
For turbidimetric assay, M. lysodeikticus cells were used as substrate for HEWL and chloroform treated E. coli B cells for T4L.
lysoplate assay is present in a soluble form and is easily degraded by active lysozyme in contrast to the turbidimetric assay in which the charge−charge interaction is the rate-limiting step. The hydrolytic activity of the immobilized lysozymes against glycol chitin, determined from ΔA420nm corresponding to that in the calibration curve obtained with different concentrations of the free enzymes (Figure S5, Supporting Information), was even higher (Table 3). These results are in line with the earlier observations with acetylated HEWL that lost its lytic activity but retained its hydrolytic activity against glycol chitin55 and provide further support for the importance of charge−charge 1604
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Table 4. Minimum Inhibitory Concentrations (MIC; μg/mL) and Minimum Bactericidal Concentrations (MBC; μg/mL) of Free and Immobilized HEW and T4 Lysozymes against Different Bacteria after 24 h at 37 °C (or 30 °C) in Brain Heart Infusion Broth M. lysodeikticus MIC CNC blanka HEWL Preparation free adsorbed (pH 7.4) coupled to EDC activated CNC coupled to Am-CNC T4L Preparation free adsorbed (pH 7.4) coupled to EDC activated CNC coupled to Am-CNC
b
MBC
Corynebacterium sp. c
E. coli
Ps. mendocina
MIC
MBC
MIC
MBC
MIC
MBC
>1250
>1250
>1250
>1250
>1250
>1250
>1250
>1250
200 300 250 125
500 1000 800 500
375 600 450 225
800 1000 1000 650
>1250 >1250 >1250 650
>1250 >1250 >1250 1000
>1250 >1250 >1250 800
>1250 >1250 >1250 1250
100 375 250 62.5
250 600 400 175
275 450 350 100
650 800 600 225
750 >1250 >1250 500
>1250 >1250 >1250 750
1000 >1250 >1250 625
>1250 >1250 >1250 1000
a Cellulose nanocrystals for different immobilization methods were used as blanks. bMIC (μg/mL) is the lowest concentration of free or immobilized lysozyme preparations that inhibited the growth of the test microorganism (no visible growth at the end of the experiment; OD620nm of the test well equal to the OD620nm of the blank). cMBC (μg/mL) is the lowest concentration of the lysozyme preparations that killed 99.9% of the test inoculum.
Figure 2. Time-kill study with Alamar Blue assay for (a) free HEWL, (b) Am-CNC-HEWL, (c) free T4L, and (d) Am-CNC-T4L against M. lysodeikticus, Corynebacterium sp., E. coli, and Ps. mendocina. After treating the bacterial cultures for 24 h with free and immobilized lysozymes, 100 μL samples were taken at different time intervals, mixed with 1× AB, incubated for 4 h at 37 °C (or 30 °C), and the absorbance was read at 560 and 595 nm.
Pristine CNC corresponding to the respective immobilized lysozyme preparations showed no antibacterial activity against any of the test organisms at a concentration of 1250 μg/mL (the highest concentration tested). Also, free HEWL did not exhibit any antibacterial activity against Gram-negative organisms as expected, while free T4L showed only bacteriostatic effect against those strains. In case of immobilized HEWL, adsorbed and covalently coupled to EDC-activated CNC preparations exhibited no bactericidal activity against Gram-negative bacteria and showed higher MIC and MBC values against the Gram-positive ones.
interaction for the lysis of bacterial cell wall that is distinct from the hydrolytic activity of the lysozymes. Antibacterial Activity of the Immobilized Lysozymes. Different assays were also used for determining the antibacterial activity of the enzyme preparations against a number of Grampositive (M. lysodeikticus and Corynebacterium sp.) and negative (E. coli and Ps. mendocina) test organisms. MIC and MBC of all enzyme preparations were determined by broth microdilution checkerboard assay in BHI broth at 37 °C (or 30 °C) for 24 h (Table 4). 1605
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Figure 3. LIVE/DEAD (Baclight) staining of bacteria treated with free and immobilized lysozyme preparations, respectively. Stained bacterial cells were visualized with confocal laser scanning microscopy (live/dead-green/red).
On the other hand, HEWL bound to Am-CNC was bactericidal against all test organisms, albeit at a higher concentration for the Gram-negatives. More or less, similar observation was made for the immobilized T4L preparations but with significantly lower MIC and MBC values than HEWL preparations. These observations are significant in terms of not only enhancing the antibacterial activity of the lysozymes by immobilization but also expanding the scope of their activity against a wider range of bacteria. The antibacterial activity of the preparations was in the order from highest to lowest: Lysozyme coupled to AmCNC > free lysozyme > lysozyme coupled to EDC activated CNC > lysozyme adsorbed to CNC (Table 4). Earlier reports on immobilized lysozyme have invariably dealt with HEWL, but characterization of their antibacterial activity is often not reported. Moreover, difference in organisms or experimental conditions used make it difficult to compare the obtained activities between different reports. However, based on all the information available so far, it seems that Am-CNC bound lysozymes displayed the lowest MIC value, for example, 650 μg/mL for HEWL bound to Am-CNC against E. coli (Table 4) in contrast to >1000 μg/mL for HEWL covalently coupled to BSA-CNC reported earlier.56 The time-kill study with Alamar Blue (AB) as an indicator for cell viability confirmed the above results. It showed the higher bactericidal activity of T4L than HEWL, for example, M. lysodeikticus cells were killed after 12 h of exposure to free T4L as compared to 16 h for free HEWL (Figure 2a,c). The test confirmed the lack of bactericidal activity of the free lysozymes against Gram-negative bacteria, and the enhanced rate of bactericidal action of both Am-CNC-lysozymes against Grampositive and -negative bacteria (Figure 2b,d); Am-CNC-T4L showed the highest rate of killing effect against M. lysodeikticus, resulting in total loss of viability within 8 h (Figure 2d). Different modification strategies have earlier been employed to extend the spectrum of HEWL antibacterial activity to
include Gram-negative bacteria, for example, by attachment of hydrophobic peptide residues to its C-terminus,57 covalent binding to fatty acids58 or binding to ZnO nanoparticles.26 In the case of ZnO-lysozyme nanoparticles, the activity is a synergism of antibacterial actions of both ZnO and the enzyme. On the other hand, while pristine Am-CNC did not display any antibacterial effect at the concentrations used but instead improved the rate and extent of bactericidal action of the immobilized lysozymes. Analysis of the cell viability of the test microorganisms, native as well as those treated with free lysozyme, Am-CNC-HEWL and Am-CNC-T4L, by staining using LIVE/DEAD kit and subsequent visualization by confocal laser scanning microscopy showed intact cells with green fluorescence, while cells with damaged cell envelope had yellow-red fluorescence (Figure 3). Most damaged cells are clearly seen for the test organisms treated with Am-CNC-lysozyme conjugates (Figure 3). Further characterization of the E. coli, Ps. mendocina, and Corynebacterium sp. cells with TEM revealed altered cell membrane morphology and cell debris with cytoplasmic aggregates after treatment with Am-CNC-lysozyme, while pristine Am-CNC shows no bactericidal effect (Figure 4). TEM images show significant changes in the majority of the treated bacterial cells, and also damaged cells with attached AmCNC-lysozyme conjugates (Figure 4b,d). The cell wall of Gram-positive bacteria is seen to be disrupted, leaving the interior of the cells as amorphous biomass (Figure 4f).
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CONCLUSIONS This report demonstrates clearly that tailored nanostructures based on an abundant renewable resource, cellulose, and a lytic enzyme could serve as highly efficient, selective, and antibacterial agents. In comparison to many other nanoparticles in use, they possess low toxicity, high biocompatibility, and selectivity. The combination of the positive charge on the 1606
DOI: 10.1021/acs.biomac.7b00219 Biomacromolecules 2017, 18, 1600−1608
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for this work. All authors discussed the results and commented on the manuscript. Funding
Financial support from the Swedish Research Council (Vetenskapsrådet, Grant No. 2016−05898) to R.H.K. and Erasmus Mundus Action 2 Welcome partnership (Application Number: WELC1104118) to A.A. is gratefully acknowledged. Notes
The authors declare no competing financial interest.
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Figure 4. TEM images of different microorganisms treated with pristine CNC and T4L immobilized to aminated CNC, respectively. (a) E. coli cells treated with pristine Am-CNC, (b) E. coli cells treated with Am-CNC-T4L, (c) Ps. mendocina cells treated with pristine AmCNC, (d) Ps. mendocina cells treated with Am-CNC-T4L, (e) Corynebacterium sp. cells treated with pristine Am-CNC, and (f) Corynebacterium sp. cells treated with Am-CNC-T4L. The scale bar is 500 nm.
nanocrystals and the lytic activity of the lysozymes in the conjugates enabled not only improving the antibacterial action but also in broadening the scope to include even Gram-negative bacteria that are normally more challenging to inactivate. Further work will involve testing of the antibacterial effect of the promising immobilized preparations on pathogenic microbes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b00219. Description of assays for determination of enzymatic activity of free- and immobilized lysozymes, assays for evaluation of the antibacterial activity of free- and immobilized lysozymes, Table S1, and Figures S1−S5 (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Phone: +46-46-222 4840. Fax: +46-46-222 4713. E-mail: rajni.
[email protected]. ORCID
Adel Abouhmad: 0000-0003-1882-5820 Tarek Dishisha: 0000-0001-8644-2640 Magdy A. Amin: 0000-0002-9292-7115 Rajni Hatti-Kaul: 0000-0001-5229-5814 Author Contributions
R.H.K. and T.D. conceived the idea. A.A. designed the experiments, analyzed the results, and wrote the first draft of the manuscript. R.H.K. and T.D. were involved in defining the orientation of the work and edited the manuscript. R.H.K. also provided the working space, laboratory facilities, and resources 1607
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