Immobilization to Positively Charged Cellulose Nanocrystals

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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, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00219 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 28, 2017

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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*,§ §

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

ACS Paragon Plus Environment

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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 Gram-positive 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 °C and 22 °C.

KEYWORDS Cellulose nanocrystals; Lysozymes; Immobilization; Lytic activity; Antibacterial activity; Zeta potential


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INTRODUCTION The concern over 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 therapies1. 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 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 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 non-selective damage to mammalian cells.3,8-9 This has rekindled interest in 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, etc.12 The attractive features of CNC are their renewable origin, low cost, ease of preparation and modification, biocompatibility, biodegradability, high mechanical and chemical strength, among others.13 Moreover, the high stability of CNC against proteolytic


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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, e.g. 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 hydrolyse 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 nanotubes25, chitosan nanofibers26, gold nanowires27, 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 wound-dressing 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 Grampositive bacteria.37


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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.

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),

N-hydroxysulfosuccinimide

(NHS),

1-ethyl-3-[3-(dimethylamino)

propyl]

carbodiimide (EDC) and lyophilized Micrococcus lysodeikticus cells were obtained from SigmaAldrich (St Louis, MO, USA). 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, USA), 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 liter of 1 M APS solution (pH 2) and heated to 60 °C for 16 h with stirring. The suspension was centrifuged (15 000 g, 10 min, 4 °C; Sorvall Lynx 4000 centrifuge, Thermo Scientific, Waltham, MA, USA), the supernatant was decanted, and the pellet of nanocrystals was washed repeatedly with deionized water with intermittent centrifugation until


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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 homogenous dispersion. To obtain CNC as ammonium salt (CNC-COO-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-COO-NH4 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 meta-periodate, 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 (15 000 g, 10 min, 4 °C), dialyzed against water using 12 000 - 14 000 molecular weight cut off dialysis membrane (Spectra/Pro 4, Spectrum Laboratories, Rancho Dominguez, CA, USA) 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). Two microliter 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-


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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, UK) was applied for determination of hydrodynamic size distribution of CNC. One-milliliter 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 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 Fifty milligrams of 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


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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 One gram 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 N-NHS and stirred at room temperature for 3 h. The activated preparation was collected by centrifugation (15 000 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. Two hundred milligrams of 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 manufacturer´s instructions.


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Immobilization yield (%) =

Total amount of protein before binding – Amount of residual protein after binding × 100 Total amount of protein before binding

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) =

Amount of immobilized protein(mg) Weight 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 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 to 10 using 10 mM of sodium acetate buffer pH 4-5, sodium phosphate buffer pH 6-7.4, or Tris-HCl buffer pH 9-10. The effect of temperature was tested through incubation of free HEWL (1 µg) and -T4L (3 µg), and CNClysozyme 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


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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.

RESULTS AND DISCUSSION Preparation and characterization of CNC and CNC-lysozyme 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 yield

38-39,41-42

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


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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 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


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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 EDCactivated 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 nanocrystals28, electrospun chitosan nanofibres50, ZnO51, TiO252, polystyrene6 and silica53 showed that significantly higher amounts of the protein could be loaded on the CNC preparations in this study. Table 1. Immobilization of hen egg white- and T4 lysozymes by different mechanisms to cellulose nanocrystals Immobilization Immobilization Amount methoda yield protein immobilized (%) a (mg/g CNC)

Total Activity protein (U/mg immobilized CNC) b (mg)

Specific Activity c

Retained activity (%) d

Physical adsorption to CNC at pH 7.4 HEWL

88

211.2

10.6

46.3×103

9.8×103

24.5

T4L

94

112.8

5.6

38.0×103

4.3×103

0.5×10-2

Covalent coupling to EDC-activated CNC HEWL

100

240.0

12.0

25.0×103

6.0×103

15

T4L

100

120.0

6.0

0.0

0.0

0.0

Covalent coupling to Am-CNC


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HEWL

96

230.4

11.5

149.8×103 34.5×103

86.3

T4L

92

110.4

5.5

58.8×108

78.3

a

6.5×108

HEWL (12 mg) and T4L (6 mg) were mixed with 50 mg CNC.

b

Enzymatic 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. c

Activity units per mg immobilized enzyme. Specific activities of free HEWL and T4L were 40 ×103 and 8.3 ×108 U per mg enzyme; d

Activity of enzyme after binding/activity of the enzyme before binding with turbidimetric assay under the same conditions. Results in the table are average of triplicates.

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, aminofunctionalization of the CNCs resulted in a 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-CNClysozymes 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


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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). Table 2. Zeta potential (ζ) in millivolt (mV) of CNC and CNC with immobilized lysozymes Samples

Zeta potential (mV)

CNC

- 54.0 ±3.0

CNC-COONH4+

- 42.0 ±0.9

Am-CNC

+ 31.0 ±1.2

HEWL

+ 9.5 ±1.6

T4L

+ 6.8 ±1.4

CNC with adsorbed HEWL

- 31.0 ±1.3

CNC with adsorbed T4L

- 32.4 ±4.0

Am-CNC with bound HEWL

+ 37.2 ±1.1

Am-CNC with bound T4L

+ 42.6 ±2.4

M. lysodeikticus cells

- 26.0 ±2.2

Chloroform treated E. coli B cells

- 21.0 ±0.8

The pH profiles of the immobilized lysozymes showed a shift in the optimum pH of the enzymatic activity towards 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.


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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 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 °C 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 1 a-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).


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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. 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


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lysoplate assay. This is because the peptidoglycan layer of the M. lysodeikticus cells in the 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 ∆A420nm 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 chitin,55 and provide further support for the importance of charge-charge interaction for the lysis of bacterial cell wall that is distinct from the hydrolytic activity of the lysozymes. Table 3. Retained activities of free- and immobilized HEW- and T4 lysozymes measured by different assays % Retained activity determined by Enzyme preparation

Turbidimetric assaya

Lysoplate assay

Glycol Chitin

Free enzyme

100.0

100.0

100.0

Adsorbed (pH7.4)

24.5

88.8 ± 1.3

92.0 ± 2.2

Coupled to EDC activated CNC

15.0 ± 3.1

90.3 ± 1.7

95.0 ± 0.7

Coupled to Am-CNC

86.3 ± 1.5

97.4 ± 2.1

98.0 ± 0.4

Free enzyme

100.0

100.0

100.0

Adsorbed (pH7.4)

0.5×10-2

60.0 ± 1.6

72 ± 2.7

Coupled to EDC-activated CNC

0.0

86.0 ± 0.4

89 ± 0.5

Coupled to Am-CNC

78.3 ± 2.8

98.0 ± 1.8

95 ± 2.1

HEWL

T4L

a

For turbidimetric assay, M. lysodeikticus cells were used as substrate for HEWL and


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chloroform treated E. coli B cells for T4L.

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). 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. 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

Corynebacterium sp. E. coli

MICb

MIC

MBC

MIC

>1250 >1250

>1250

>1250

>1250 >1250 >1250 >1250

Free

200

500

375

800

>1250 >1250 >1250 >1250

Adsorbed (pH 7.4)

300

1000

600

1000

>1250 >1250 >1250 >1250


250

800

450

1000

>1250 >1250 >1250 >1250

Coupled to Am-CNC

125

500

225

650

650

CNC blank a

MBCc

Ps. mendocina MBC

MIC

MBC

HEWL preparation

1000

800

1250


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T4L preparation Free

100

250

275

650

750

Adsorbed (pH7.4)

375

600

450

800

>1250 >1250 >1250 >1250


250

400

350

600

>1250 >1250 >1250 >1250

Coupled to Am-CNC

62.5

175

100

225

500

a

>1250 1000

750

625

>1250

1000

Cellulose nanocrystals for different immobilization methods were used as blanks.

b

MIC (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). c

MBC (µg/ml) is the lowest concentration of the lysozyme preparations that killed 99.9% of the test inoculum.

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. 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 Am-CNC > 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


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so far, it seems that Am-CNC bound lysozymes displayed the lowest MIC value, e.g. 650 µg/ml for HEWL bound to 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 ALAMARBLUE® (AB) as an indicator for cell viability confirmed the above results. It showed the higher bactericidal activity of T4L than HEWL, e.g. M. lysodeikticus cells were killed after 12 hours of exposure to free T4L as compared to 16 hours 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 Gram-positive 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 2 d).

Figure 2. Time-kill study with ALAMARBLUE

®

assay for a) free HEWL, b) Am-CNC-

HEWL, c) free T4L and d) Am-CNC-T4L against M. lysodeikticus, Corynebacterium sp., E. coli


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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 1X AB, incubated for 4 h at 37 °C (or 30 °C) and the absorbance was read at 560 and 595 nm. Different modification strategies have earlier been employed to extend the spectrum of HEWL antibacterial activity to include Gram-negative bacteria e.g. by attachment of hydrophobic peptide residues to its C-terminus,57 covalent binding to fatty acids,58 or binding to ZnO nanoparticles.26 In 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).


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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). 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 Am-CNC-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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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 Am-CNC, (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.

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, 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 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


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more challenging to inactivate. Further work will involve testing of the antibacterial effect of the promising immobilized preparations on pathogenic microbes.

ASSOCIATED CONTENT Supporting Information. 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, S2, S3, S4 and S5.

AUTHOR INFORMATION Corresponding Author * Address: Biotechnology, Department of Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: +46-46-222 4840; Fax: int+46-46-222 4713; E-mail: [email protected] ORCID Rajni Hatti-Kaul: 0000-0001-5229-5814 Adel Abouhmad: 0000-0003-1882-5820 Tarek Dishisha: 0000-0001-8644-2640 Magdy Amin: 0000-0002-9292-7115

Author Contributions


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RHK and TD conceived the idea. AA designed the experiments, analyzed the results and wrote the first draft of the manuscript. RHK and TD were involved in defining the orientation of the work, and edited the manuscript. RHK also provided the working space, laboratory facilities and resources for this work. All authors discussed the results and commented on the manuscript. Funding Sources Financial support from the Swedish Research Council (Vetenskapsrådet, grant no. 2016-05898) to RHK and Erasmus Mundus Action 2 Welcome partnership (application number: WELC1104118) to AA is gratefully acknowledged. Notes The authors declare no competing financial interest.

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TABLE OF CONTENTS


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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Immobilization to Positively Charged Cellulose Nanocrystals

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