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Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles
Microbiological Research 178 (2015) 35–41
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Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles Yulong Tan a,b,c,1 , Su Ma a,b,c,1 , Chenguang Liu d , Wengong Yu a,b,c , Feng Han a,b,c,∗ a
Key Laboratory of Marine Drugs, Chinese Ministry of Education, Qingdao, China Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Qingdao, China School of Medicine and Pharmacy, Ocean University of China, Qingdao, China d College of Marine Life Sciences, Ocean University of China, Qingdao, China b c
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 18 May 2015 Accepted 1 June 2015 Available online 14 June 2015 Keywords: Immobilization Stability Biofilm Nanoparticles Carboxymethyl chitosan
a b s t r a c t A -N-acetyl-glucosaminidase (DspB) from Aggregatibacter actinomycetemcomitans CU1000 has been proved to inhibit and detach the biofilms formed by Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans. However, the application of this enzyme is limited by its poor stability. In the present study, a -N-acetyl-glucosaminidase encoding gene, dspB, was cloned from A. actinomycetemcomitans HK1651 and expressed in Escherichia coli. The recombinant DspB was loaded on hydrogel nanoparticles, which was prepared by using linoleic acid (LA) modified carboxymethyl chitosan (CMCS) after sonication. The nanoparticles were almost saturated by DspB at 0.3 mg/ml, which gave a loading capacity of 76.7%. The immobilization enhanced thermal stability, storage stability and reusability of DspB significantly. Moreover, it also increased antibiofilm activity due to the dual mechanism, including the improvement of the enzyme stability and the antibiofilm activity of CMCS nanoparticles. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Biofilms are structured, specialized communities of adherent microorganisms encased in a complex extrapolymeric substance (EPS) (Rohde et al. 2010). The EPS provides mechanical support, mediates cell–cell and cell–surface interactions, and acts as protective environment (Alves and Pereira 2014). Bacterial cells within biofilms were found to be 1000-fold less susceptible to diverse antibiotics than planktonic ones (Swidsinski et al. 2008). Therefore, biofilm formations on medical devices, such as implant surface, significantly increase the risk of infection. The mature biofilms are difficult to be removed by chemical (antibacterial agents) or physical (heating, sonication or vortexing) treatments (Fine et al. 1999; Wolcott et al. 2013). Recently, the use of enzymes which remove biofilm by destroying the EPS has become an attractive strategy for the control and removal of biofilms (Badel et al. 2008; Augustine et al. 2010; Lamppa et al. 2011; Elchinger et al. 2014). Kaplan et al. has identified a -N-acetyl-glucosaminidase, DspB, from Aggregatibacter
∗ Corresponding author at: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. Tel.: +86 532 82032067; fax: +86 532 82033054. E-mail address: [email protected] (F. Han). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.micres.2015.06.001 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
actinomycetemcomitans CU1000 (formerly named as Actinobacillus actinomycetemcomitans CU1000) that degraded the poly-(1,6)-Nacetyl-glucosamine (PNAG), which is the major polysaccharide in EPS of Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans (Kaplan et al. 2004). It has been shown that DspB caused disaggregation and detachment of biofilms formed by several bacterial species (Brindle et al. 2011; Kaplan et al. 2011). However, the application of this enzyme is limited by its instability. Immobilization of enzymes usually greatly improves their stability and reusability, therefore has attracted more and more attention (Sathishkumar et al. 2012; Twala et al. 2012; Chauhan et al. 2013). Chitosan and its derivatives can form hydrogel nanoparticles in water through self-aggregation (Tan and Liu 2011; Jin et al. 2012), which are one of the most suitable matrix for enzyme immobilization because of a higher specific surface area for loading a larger amount of enzyme, nontoxicity and biocompatibility (Tan et al. 2008; Kuo et al. 2012). Moreover, antibiofilm activities of chitosan and some of its derivatives also have been well described (Martinez et al. 2010; Orgaz et al. 2011; Zhang et al. 2013). In our previous study, a chitosan derivative, carboxymethyl chitosan (CMCS), could inhibit biofilm formation through the dual mechanism including aggregation by charge neutralization and flocculation by bridging mechanism (Tan et al. 2011). In this study, DspB from A. actinomycetemcomitans HK1651 was immobilized on CMCS nanoparticles. The thermal stability, storage
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stability and reusability of immobilized enzyme were evaluated and compared with the free enzyme. Moreover, the antibiofilm efficacy of immobilized enzyme was evaluated towards S. aureus, S. epidermidis and A. actinomycetemcomitans, and the potential mechanisms were discussed.
2. Materials and methods 2.1. Bacterial strains, media and reagents The bacterial strains used in this study were S. aureus RN6390, 15981, 8325 and Col, S. epidermidis QY301, RP62A, M187 and 1457, A. actinomycetemcomitans HK1651. These strains were cultured in Tryptic Soy Broth (TSB) medium at 37 ◦ C. CMCS (deacetylation degree = 90% and viscosity = 80 mPa s) was obtained from Oddfoni Chemical Co. (Nanjing, China). The O-carboxymethylation degree, further calculated based on 1 H NMR spectrum, was 89%. Linoleic acid (LA), 1-ethyl-3-(3dimethylaminopropyyl) carbodiimide (EDC) and 4-nitrophenyl-Nacetyl -D-glucosaminide were purchased from Sigma Chemicals. All other chemicals used in this study were of analytical grade.
2.2. Expression and purification of DspB To express DspB protein, the primers pDspB-F: 5 -GGAATTCCATATGAATTGTTGCGTAAAAGG (the Nde I site is underlined) and pDspB-R: 5 -CCGCTCGAGCTCATCCCCATTCGTCTTAT (the Xho I site is underlined) were designed to amplify the dspB gene using the genomic DNA of A. actinomycetemcomitans HK1651 as the template. An expected PCR product of 1.1 kb was purified from the agarose gels, digested with Nde I and Xho I, and ligated into a similarly digested pET-24a (+). The resulting pET-24a-DspB was transformed into E. coli BL21 (DE3) competent cells. E. coli BL21 (DE3) cells containing plasmid pET-24a-DspB were grown at 37 ◦ C in LB medium supplemented with kanamycin (30 g/ml) until the optical density at 600 nm was about 0.5, then isopropyl--d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After incubation at 25 ◦ C for an additional 24 h, the cells were harvested by centrifugation (6000 × g, 10 min), resuspended in buffer A (20 mM phosphate buffer, pH 7.0/500 mM NaCl), and disrupted by ultrasonication. After centrifugation (10,000 × g, 30 min), the supernatants were loaded to an affinity column (HisTrap HP 5 ml, GE health) and eluted by a linear gradient of imidazole (5–500 mM) in buffer A. -1,6-Nacetylglucosaminidase activity containing fractions were pooled, dialyzed against phosphate buffer (20 mM, pH 6.0) and stored at −20 ◦ C. SDS-PAGE was performed to detect the purified enzyme as described by Laemmli (Laemmli 1970). Protein concentration was determined using BCA protein assay reagent (Pierce Biotechnology, USA) using bovine serum albumin as the standard protein.
2.3. Enzymatic activity assay The activities of free and immobilized DspB were assayed using 4-nitrophenyl-N-acetyl -D-glucosaminide as the substrate. Ten microliter of DspB was added to 0.2 ml of substrate solution [5 mM substrate in 50 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl] and incubated at 40 ◦ C for 10 min. The reactions were terminated by adding 5 l of 10 M NaOH. The increase in absorption resulting from the release of p-nitrophenolate was measured with a spectrophotometer set to 405 nm. One unit (U) of the enzyme activity was defined as the amount of enzyme that produces 1 mol p-nitrophenolate per min under the above conditions.
2.4. Immobilization of DspB on LA-CMCS nanoparticles Hydrogel nanoparticles were prepared using LA modified CMCS as described in previous study (Liu et al. 2007). The DspB was added to 1 ml of nanoparticles (0.20 mg/ml) to a final concentration of 0.05–0.40 mg/ml, and then the suspension was sonicated. The immobilized enzyme was obtained by ultrafiltration with the Centricon centrifugal filter devices (molecular weight cut-off 50 kDa, Millipore). The filtrate (unloaded DspB) was collected to calculate the residual amount of protein. The DspB loading capacity (LC) of nanoparticles and DspB loading efficiency (LE) were calculated using the following equations: LC =
(A − B) C
LE % =
(A − B) × 100 A
A = Total amount of added DspB; B = Total amount of unloaded DspB; C = Weight of the nanoparticles measured after freeze-dry. The assays were performed in triplicate on separate experiments. The data collected in this study were expressed as the mean value ± standard deviation (SD). 2.5. The effect of temperature and pH on free and immobilized enzymes The optimum temperature of the free and immobilized enzyme activity was determined by carrying out the enzyme activity assay in phosphate buffer (pH 6.0) at temperatures ranging from 20 ◦ C to 80 ◦ C. The effect of pH on the activity of the free and immobilized enzyme was determined under the standard assay conditions at 40 ◦ C in various buffers (50 mM): citrate acid buffer (pH 4.0–5.6), phosphate buffer (pH 5.6–8.0). All assays were performed at least in triplicate. 2.6. Stability and reusability of free and immobilized enzymes The thermal stability of free and immobilized DspB was evaluated by measuring the residual activities of DspB after incubation at various temperatures at pH 6.0 for 1 h. The storage stability of free and immobilized DspB was investigated by measuring the residual activities after being stored at 37 ◦ C for various times. The reusability of the immobilized DspB was determined in a repeated catalysis process. After each reaction, the used enzyme was washed with phosphate buffer (pH 6.0) for the next use and then added to a substrate solution to start a new cycle. Activities found for each repetition were compared with the initial activity assuming as 100%. 2.7. Biofilm assay Biofilm formation in 96-well microtiter plates was conducted as described previously (Chauhan et al. 2013). In brief, bacteria from overnight cultures were diluted to 1 × 106 colony-forming units (CFU)/ml. Each well (Costar, Cambridge, MA, USA) was filled with 100 l aliquots of the diluted culture. The plates were incubated at 37 ◦ C for 24 h without shaking, and washed three times with 0.9% (w/v) NaCl. Biofilms were stained with 150 l of 0.1% (w/v) crystal violet solution for 30 min, washed twice with 0.9% (w/v) NaCl and incubated in 200 l of 30% (v/v) acetic acid for 15 min to extract the crystal violet retained by the cells. The extract was used to determine the amount of biofilm by measuring its A590 with a microtiter plate reader (DynaTech, Chantilly, Va.). For inhibition studies, TSB medium containing DspB at different concentrations was used to dilute the overnight culture of bacteria. For detachment studies, biofilms were formed using non-DspB TSB medium, and then treated by DspB at different concentrations for 1 h. At least six replicates were conducted for each sample, and each experiment was performed at least in triplicate.
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0.30 mg/ml. When the enzyme concentration was high enough, the nanoparticles were almost saturated due to the fixed amount of enzymes immobilized on the surface of the nanoparticles (Gan et al. 2012). 3.2. Effect of temperature and pH on activity of free and immobilized DspB The activity of the free and immobilized DspB increased up to 40 ◦ C and then decreased with further increases in temperature. The optimum temperature for the free and immobilized DspB was 40 ◦ C (Fig. 2 A). And the optimum pH was pH 6.0 for both enzymatic forms (Fig. 2B). Therefore, immobilization of DspB on CMCS nanoparticle does not change its optimum temperature and pH. Fig. 1. Transmission electron micrograph of nanoparticles. The samples were immobilized on copper grids and dried at room temperature. Then they were stained with phosphate tungsten acid and examined by TEM.
3. Results and discussion 3.1. Determination of DspB loading capacity and efficiency The size and morphology of the nanoparticles were observed by transmission electron microscopy (TEM) (Fig. 1). TEM images showed that nanoparticles have nearly spherical shape and smooth surface. And the size range of the nanoparticles was between 100 and 500 nm. The loading capacity and efficiency of DspB were affected by its concentration (Table 1). As the concentration of DspB increased in the range 0.05–0.40 mg/ml, the loading efficiency decreased from 79.25 ± 1.50% to 39.52 ± 0.08%. On the other hand, the DspB loading capacity was enhanced dramatically from 198.13 ± 3.75 mg/g to 767.08 ± 13.90 mg/g by increasing the DspB concentration from 0.05 to 0.30 mg/ml. However, the loading capacity did not increase significantly from 0.30 to 0.40 mg/ml, indicating that nanoparticles were almost saturated by DspB at
3.3. Stability of free and immobilized DspB 3.3.1. Thermal stability of free and immobilized DspB The effect of temperature on the stability of free and immobilized DspB at pH 6.0 was shown in Fig. 3. The activities of both free and immobilized DspB were not adversely affected at temperatures below 40 ◦ C. The residual activity dropped significantly above 40 ◦ C. However, compared with free enzyme, immobilized DspB lost less activity and retained 21.0% of its initial activity after incubation at 70 ◦ C for 60 min, whereas the free DspB lost almost all activity. The improved thermal stability for the nanoparticle-coupled enzyme is likely because some enzyme molecules have been entrapped into the core of nanoparticles, which could provide more rigid external backbone for enzyme molecules and protect
Table 1 DspB loading capacity (LC) and loading efficiency (LE). DspB (mg)
LE (%)
0.05 0.10 0.20 0.30 0.40
79.25 66.42 59.52 51.14 39.52
LC (mg/g) ± ± ± ± ±
1.50 0.07 1.84 0.93 0.08
198.13 332.08 595.21 767.08 790.42
± ± ± ± ±
3.75 0.36 18.44 13.90 1.56
Fig. 3. Thermal stability of free (open triangles) and immobilized (closed triangles) DspB. The enzyme was incubated in 50 mM phosphate buffer (pH 6.0) without substrate for 1 h at 0–70 ◦ C, respectively. Residual activities were measured at 40 ◦ C and pH 6.0. The activity of enzyme without incubation was assigned a value of 100%.
Fig. 2. Effect of temperature (A) and pH (B) on the activity of free and immobilized DspB. (A) The optimal temperature of free (open triangles) and immobilized (closed triangles) DspB was determined by measuring the activity in 50 mM sodium phosphate buffer (pH 6.0) at various temperatures (20–80 ◦ C). (B) The optimal pH of free (open triangles and circles) and immobilized (closed triangles and circles) DspB was determined by measuring the activity at 40 ◦ C in 50 mM citrate acid buffer (pH 4.0–5.6; triangles), phosphate buffer (pH 5.6–8.0; circles). The enzymatic activity of a fresh sample of free or immobilized DspB measured at pH 6.0 and 40 ◦ C was defined as 100%.
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Fig. 4. Storage stability of free (open triangles) and immobilized (closed triangles) DspB. The enzyme was incubated in 50 mM phosphate buffer (pH 6.0) without substrate at 37 ◦ C for different periods of time, respectively. Residual activities were measured at 40 ◦ C and pH 6.0. The activity of enzyme without incubation was assigned a value of 100%.
the enzymatic configuration from distortion or damage by heat exchange (Nawani et al. 2006). 3.3.2. Storage stability of free and immobilized DspB Fig. 4 showed the storage stability of the free and immobilized DspB at 37 ◦ C. The free DspB was stable for short incubation periods but was adversely affected at longer incubation time, losing its activity completely within 7 h at 37 ◦ C. In contrast, the immobilized enzyme retained 33.4% of its initial activity after 7 h. These results indicated that enzyme storage stability was significantly improved by immobilization. The increased stability of immobilized enzyme may result from multiple interactions (e.g. electristic interaction, hydrogen bonding and hydrophobic interaction) between DspB and the nanoparticles (Ren et al. 2011). Enzymes are quite sensitive against the environmental situation and may lose their activities easily, thus, the stability of immobilized enzyme is extremely important for subsequent applications, especially in vivo. 3.3.3. Reusability of immobilized DspB The reusability of DspB immobilized on CMCS nanoparticles has also been studied because of its importance for industry to reduce the processing costs. After 8 cycles, the activity of immobilized DspB maintained at a level exceeding 60% of the original activity (Fig. 5). The loss of immobilized DspB activity may be attributed to the conformational changes during reuse or the leakage from the support matrix (Keerti Gupta et al., 2014). From a commercial point of view, the reusability of enzymes is one of the most important features for the immobilization. The result showed that the immobilized enzyme had excellent operational stability. Such reusability could significantly reduce the
Fig. 5. Reusability of immobilized DspB. The used enzyme was washed with distilled water, then added to a substrate solution to start a new cycle. Activity found for each repetition was compared with the initial activity, assuming it was 100%.
operation costs in applications. Moreover, immobilized DspB on nanoparticles can be used on the surface of medical materials in vivo to prevent biofilm formation, which can greatly extend the range of application of this enzyme. The results of immobilization strongly depend on the properties of supports (Wang et al. 2009). Recently, nanotechnology has shown a significant promise in the preparation of immobilized enzymes. Nanomaterials embody distinctive physicochemical and biological properties, such as small size, magnetism and large surface area to volume ratio, which make them favorable for enzyme immobilization (Li et al. 2008; Rai et al. 2009). Immobilization of enzymes on nanoparticles can improve their resistance to denaturing compounds, stability to pH and temperature, as well as an adequate environment for their repeated use (Fang et al. 2009). In addition, the irreversible aggregation of enzyme on heating can be completely prevented by complexation between the heatdenatured enzyme and nanoparticles according to a mechanism similar to that of a molecular chaperone (Takehiro et al., 1994; Akiyoshi et al., 1999; Nomura et al., 2003). Chitosan is an attractive natural biopolymer with large primary amine groups and positive charge. It has shown excellent biocompatibility, susceptibility to chemical modifications, as well as the ability to increase membrane permeability (Colonna et al. 2008; Yang et al. 2010). Moreover, chitosan nanoparticle can easily be prepared, which is one of the most promising support material for enzyme immobilization (Wang et al. 2013; Zang et al. 2014; Long et al. 2015). It is reported that the performance of -galactosidase immobilized on chitosan nanoparticles was higher than that immobilized on chitosan micro- and macroparticles (Biro et al. 2008). This can be ascribed to the better distribution of the enzyme on the nanoparticles due to high specific surface area of chitosan nanoparticles and large of active functional groups available for fixing the enzyme molecules (Tang et al. 2006).
3.4. Antibiofilm activity of free and immobilized DspB 3.4.1. Inhibition of biofilm formation by free and immobilized DspB Biofilm formation of S. epidermidis RP62A was significantly inhibited by both free and immobilized enzymes in a concentration-dependent manner (Fig. 6). Immobilized DspB showed greater antibiofilm activity than free enzyme. When the concentration of DspB is 0.30 U/ml, 81.5% of biofilm formation of S. epidermidis RP62A was inhibited by free enzymes and 93.8% by immobilized enzyme. To determine the antibiofilm activity spectrum of free and immobilized DspB, the effects of DspB on biofilm formations of various strains (S. aureus, S. epidermidis and A. actinomycetemcomitans) were measured using 96-well microtiter plate assay. The strains were cultured in the presence of 0.30 U/ml DspB for 24 h to form biofilms. As shown in Fig. 7 A, biofilm formations by all of 9 strains were inhibited by both free and immobilized DspB. Immobilized DspB exhibited stronger inhibition effect (>90%) than free DspB (≈80%). Interestingly, CMCS nanoparticles also exhibited prevention effect on biofilm formation (from 6% to 50% at 0.156 to 2.500 mg/ml). In our previous study, CMCS prevented initial bacterial adherence and cell–cell interaction, which is involved in the aggregation through charge neutralization and flocculation by bridging mechanism. CMCS nanoparticles does not affect the cationic charge, but also exhibited a certain antibiofilm activities. Additionally, the immobilized enzyme enhanced stability of DspB, which increased biofilm formation inhibition effect of DspB. These results together may explain the mechanism of the increased biofilm formation inhibition effect of immobilized DspB.
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Fig. 6. Inhibition of S. epidermidis RP62A biofilm formation by nanoparticles, free and immobilized DspB in 96-well microtiter plates. (A) The strains from an overnight culture were diluted to 1 × 106 colony-forming units (CFU)/ml with TSB media containing DspB at different concentrations (range 0–0.3 U/ml). After 24 h cultivation, wells were rinsed and stained with crystal violet. (B) Quantitation of crystal violet staining in panel A. Wells were destained with acetic acid and the absorbance of the crystal violet solution was measured at 590 nm. Absorbance is proportional to biofilm biomass.
Fig. 7. Inhibition (A) and detachment (B) effect of nanoparticles, free and immobilized DspB on biofilm formations in 96-well microtiter plates. For inhibition studies, TSB medium containing 0.3 U/ml DspB was used. For detachment studies, bacteria were cultured in non-DspB TSB medium for 24 h to form mature biofilm, then treated with 0.3 U/ml DspB for 1 h. Wells were stained with crystal violet and destained with acetic acid. The absorbance of the crystal violet solution was measured at 590 nm. Absorbance is proportional to biofilm biomass.
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3.4.2. Detachment of mature biofilm by free and immobilized DspB The detachment effect of DspB on mature biofilms was also explored. The strains were cultured for 24 h to form mature biofilms, and then biofilms were treated by DspB for 1 h. As shown in Fig. 7B, both free and immobilized DspB disrupted the preexisting biofilms of all 9 strains. Immobilized DspB showed similar effect with free DspB (≈80%), which is ascribed to the comparable activity of free and immobilized enzymes. 4. Conclusion In this work, DspB was loaded on nanoparticles of LA-CMCS. The immobilization enhanced thermal stability, storage stability and reusability of DspB significantly. The immobilized enzyme was more effective than free enzyme in inhibiting and detaching the biofilm formations by several bacterial species such as S. aureus, S. epidermidis and A. actinomycetemcomitans. The studies also show that CMCS nanoparticles can exhibit prevention effect on biofilm formation, which can be used as a kind of excellent delivery of antibiofilm agents. These results suggest that DspB immobilized on CMCS nanoparticles can be used for treatment of medical device-associated infections. Moreover, the complex of DspB and nanoparticles can eventually be expanded in vivo, which will further broaden its range of application. Acknowledgements This work was supported by National High-tech R&D Program (2014AA093504 and 2007AA091506), National Natural Science Foundation of China (U1406402), Science and Technology Development Project of Shandong Province (2014GGH215002). References Akiyoshi K, Sasaki Y, Sunamoto J. Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B. Bioconjugate Chem 1999;10(3): 321–4. Alves D, Pereira MO. Mini-review: antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces. Biofouling 2014;30(4): 483–99. Augustine N, Kumar P, Thomas S. Inhibition of Vibrio cholerae biofilm by AiiA enzyme produced from Bacillus spp. Arch Microbiol 2010;192(12): 1019–22. Badel S, Laroche C, Gardarin C, Bernardi T, Michaud P. New method showing the influence of matrix components in Leuconostoc mesenteroides biofilm formation. Appl Biochem Biotech 2008;151(2-3):364–70. Biro E, Nemeth AS, Sisak C, Feczko T, Gyenis J. Preparation of chitosan particles suitable for enzyme immobilization. J Biochem Biophys Methods 2008;70(6):1240–6. Brindle ER, Miller DA, Stewart PS. Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB. Biotechnol Bioeng 2011;108(12):2968–77. Chauhan N, Narang J, Sunny Pundir CS. Immobilization of lysine oxidase on a goldplatinum nanoparticles modified Au electrode for detection of lysine. Enzyme Microb Technol 2013;52(4-5):265–71. Colonna C, Conti B, Perugini P, Pavanetto F, Modena T, Dorati R, et al. Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy. Eur J Pharmaceutics Biopharmaceutics 2008;70(1): 58–65. Elchinger PH, Delattre C, Faure S, Roy O, Badel S, Bernardi T, et al. Effect of proteases against biofilms of Staphylococcus aureus and Staphylococcus epidermidis. Lett Appl Microbiol 2014;59(5):507–13. Fang H, Huang J, Ding L, Li M, Chen Z. Preparation of magnetic chitosan nanoparticles and immobilization of laccase. J Wuhan Univ Technol Mater Sci Ed 2009;24(1):42–7. Fine DH, Furgang D, Kaplan J, Charlesworth J, Figurski DH. Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite. Arch Oral Biol 1999;44(12):1063–76. Gan Z, Zhang T, Liu Y, Wu D. Temperature-triggered enzyme immobilization and release based on cross-linked gelatin nanoparticles. PloS One 2012;7(10):e47154.
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Enhancing the stability and antibiofilm activity of DspB by immobilization on carboxymethyl chitosan nanoparticles Yulong Tan a,b,c,1 , Su Ma a,b,c,1 , Chenguang Liu d , Wengong Yu a,b,c , Feng Han a,b,c,∗ a
Key Laboratory of Marine Drugs, Chinese Ministry of Education, Qingdao, China Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Qingdao, China School of Medicine and Pharmacy, Ocean University of China, Qingdao, China d College of Marine Life Sciences, Ocean University of China, Qingdao, China b c
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 18 May 2015 Accepted 1 June 2015 Available online 14 June 2015 Keywords: Immobilization Stability Biofilm Nanoparticles Carboxymethyl chitosan
a b s t r a c t A -N-acetyl-glucosaminidase (DspB) from Aggregatibacter actinomycetemcomitans CU1000 has been proved to inhibit and detach the biofilms formed by Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans. However, the application of this enzyme is limited by its poor stability. In the present study, a -N-acetyl-glucosaminidase encoding gene, dspB, was cloned from A. actinomycetemcomitans HK1651 and expressed in Escherichia coli. The recombinant DspB was loaded on hydrogel nanoparticles, which was prepared by using linoleic acid (LA) modified carboxymethyl chitosan (CMCS) after sonication. The nanoparticles were almost saturated by DspB at 0.3 mg/ml, which gave a loading capacity of 76.7%. The immobilization enhanced thermal stability, storage stability and reusability of DspB significantly. Moreover, it also increased antibiofilm activity due to the dual mechanism, including the improvement of the enzyme stability and the antibiofilm activity of CMCS nanoparticles. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction Biofilms are structured, specialized communities of adherent microorganisms encased in a complex extrapolymeric substance (EPS) (Rohde et al. 2010). The EPS provides mechanical support, mediates cell–cell and cell–surface interactions, and acts as protective environment (Alves and Pereira 2014). Bacterial cells within biofilms were found to be 1000-fold less susceptible to diverse antibiotics than planktonic ones (Swidsinski et al. 2008). Therefore, biofilm formations on medical devices, such as implant surface, significantly increase the risk of infection. The mature biofilms are difficult to be removed by chemical (antibacterial agents) or physical (heating, sonication or vortexing) treatments (Fine et al. 1999; Wolcott et al. 2013). Recently, the use of enzymes which remove biofilm by destroying the EPS has become an attractive strategy for the control and removal of biofilms (Badel et al. 2008; Augustine et al. 2010; Lamppa et al. 2011; Elchinger et al. 2014). Kaplan et al. has identified a -N-acetyl-glucosaminidase, DspB, from Aggregatibacter
∗ Corresponding author at: School of Medicine and Pharmacy, Ocean University of China, Qingdao, China. Tel.: +86 532 82032067; fax: +86 532 82033054. E-mail address: [email protected] (F. Han). 1 Both these authors contributed equally to this work. http://dx.doi.org/10.1016/j.micres.2015.06.001 0944-5013/© 2015 Elsevier GmbH. All rights reserved.
actinomycetemcomitans CU1000 (formerly named as Actinobacillus actinomycetemcomitans CU1000) that degraded the poly-(1,6)-Nacetyl-glucosamine (PNAG), which is the major polysaccharide in EPS of Staphylococcus epidermidis, Staphylococcus aureus and A. actinomycetemcomitans (Kaplan et al. 2004). It has been shown that DspB caused disaggregation and detachment of biofilms formed by several bacterial species (Brindle et al. 2011; Kaplan et al. 2011). However, the application of this enzyme is limited by its instability. Immobilization of enzymes usually greatly improves their stability and reusability, therefore has attracted more and more attention (Sathishkumar et al. 2012; Twala et al. 2012; Chauhan et al. 2013). Chitosan and its derivatives can form hydrogel nanoparticles in water through self-aggregation (Tan and Liu 2011; Jin et al. 2012), which are one of the most suitable matrix for enzyme immobilization because of a higher specific surface area for loading a larger amount of enzyme, nontoxicity and biocompatibility (Tan et al. 2008; Kuo et al. 2012). Moreover, antibiofilm activities of chitosan and some of its derivatives also have been well described (Martinez et al. 2010; Orgaz et al. 2011; Zhang et al. 2013). In our previous study, a chitosan derivative, carboxymethyl chitosan (CMCS), could inhibit biofilm formation through the dual mechanism including aggregation by charge neutralization and flocculation by bridging mechanism (Tan et al. 2011). In this study, DspB from A. actinomycetemcomitans HK1651 was immobilized on CMCS nanoparticles. The thermal stability, storage
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stability and reusability of immobilized enzyme were evaluated and compared with the free enzyme. Moreover, the antibiofilm efficacy of immobilized enzyme was evaluated towards S. aureus, S. epidermidis and A. actinomycetemcomitans, and the potential mechanisms were discussed.
2. Materials and methods 2.1. Bacterial strains, media and reagents The bacterial strains used in this study were S. aureus RN6390, 15981, 8325 and Col, S. epidermidis QY301, RP62A, M187 and 1457, A. actinomycetemcomitans HK1651. These strains were cultured in Tryptic Soy Broth (TSB) medium at 37 ◦ C. CMCS (deacetylation degree = 90% and viscosity = 80 mPa s) was obtained from Oddfoni Chemical Co. (Nanjing, China). The O-carboxymethylation degree, further calculated based on 1 H NMR spectrum, was 89%. Linoleic acid (LA), 1-ethyl-3-(3dimethylaminopropyyl) carbodiimide (EDC) and 4-nitrophenyl-Nacetyl -D-glucosaminide were purchased from Sigma Chemicals. All other chemicals used in this study were of analytical grade.
2.2. Expression and purification of DspB To express DspB protein, the primers pDspB-F: 5 -GGAATTCCATATGAATTGTTGCGTAAAAGG (the Nde I site is underlined) and pDspB-R: 5 -CCGCTCGAGCTCATCCCCATTCGTCTTAT (the Xho I site is underlined) were designed to amplify the dspB gene using the genomic DNA of A. actinomycetemcomitans HK1651 as the template. An expected PCR product of 1.1 kb was purified from the agarose gels, digested with Nde I and Xho I, and ligated into a similarly digested pET-24a (+). The resulting pET-24a-DspB was transformed into E. coli BL21 (DE3) competent cells. E. coli BL21 (DE3) cells containing plasmid pET-24a-DspB were grown at 37 ◦ C in LB medium supplemented with kanamycin (30 g/ml) until the optical density at 600 nm was about 0.5, then isopropyl--d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.2 mM. After incubation at 25 ◦ C for an additional 24 h, the cells were harvested by centrifugation (6000 × g, 10 min), resuspended in buffer A (20 mM phosphate buffer, pH 7.0/500 mM NaCl), and disrupted by ultrasonication. After centrifugation (10,000 × g, 30 min), the supernatants were loaded to an affinity column (HisTrap HP 5 ml, GE health) and eluted by a linear gradient of imidazole (5–500 mM) in buffer A. -1,6-Nacetylglucosaminidase activity containing fractions were pooled, dialyzed against phosphate buffer (20 mM, pH 6.0) and stored at −20 ◦ C. SDS-PAGE was performed to detect the purified enzyme as described by Laemmli (Laemmli 1970). Protein concentration was determined using BCA protein assay reagent (Pierce Biotechnology, USA) using bovine serum albumin as the standard protein.
2.3. Enzymatic activity assay The activities of free and immobilized DspB were assayed using 4-nitrophenyl-N-acetyl -D-glucosaminide as the substrate. Ten microliter of DspB was added to 0.2 ml of substrate solution [5 mM substrate in 50 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl] and incubated at 40 ◦ C for 10 min. The reactions were terminated by adding 5 l of 10 M NaOH. The increase in absorption resulting from the release of p-nitrophenolate was measured with a spectrophotometer set to 405 nm. One unit (U) of the enzyme activity was defined as the amount of enzyme that produces 1 mol p-nitrophenolate per min under the above conditions.
2.4. Immobilization of DspB on LA-CMCS nanoparticles Hydrogel nanoparticles were prepared using LA modified CMCS as described in previous study (Liu et al. 2007). The DspB was added to 1 ml of nanoparticles (0.20 mg/ml) to a final concentration of 0.05–0.40 mg/ml, and then the suspension was sonicated. The immobilized enzyme was obtained by ultrafiltration with the Centricon centrifugal filter devices (molecular weight cut-off 50 kDa, Millipore). The filtrate (unloaded DspB) was collected to calculate the residual amount of protein. The DspB loading capacity (LC) of nanoparticles and DspB loading efficiency (LE) were calculated using the following equations: LC =
(A − B) C
LE % =
(A − B) × 100 A
A = Total amount of added DspB; B = Total amount of unloaded DspB; C = Weight of the nanoparticles measured after freeze-dry. The assays were performed in triplicate on separate experiments. The data collected in this study were expressed as the mean value ± standard deviation (SD). 2.5. The effect of temperature and pH on free and immobilized enzymes The optimum temperature of the free and immobilized enzyme activity was determined by carrying out the enzyme activity assay in phosphate buffer (pH 6.0) at temperatures ranging from 20 ◦ C to 80 ◦ C. The effect of pH on the activity of the free and immobilized enzyme was determined under the standard assay conditions at 40 ◦ C in various buffers (50 mM): citrate acid buffer (pH 4.0–5.6), phosphate buffer (pH 5.6–8.0). All assays were performed at least in triplicate. 2.6. Stability and reusability of free and immobilized enzymes The thermal stability of free and immobilized DspB was evaluated by measuring the residual activities of DspB after incubation at various temperatures at pH 6.0 for 1 h. The storage stability of free and immobilized DspB was investigated by measuring the residual activities after being stored at 37 ◦ C for various times. The reusability of the immobilized DspB was determined in a repeated catalysis process. After each reaction, the used enzyme was washed with phosphate buffer (pH 6.0) for the next use and then added to a substrate solution to start a new cycle. Activities found for each repetition were compared with the initial activity assuming as 100%. 2.7. Biofilm assay Biofilm formation in 96-well microtiter plates was conducted as described previously (Chauhan et al. 2013). In brief, bacteria from overnight cultures were diluted to 1 × 106 colony-forming units (CFU)/ml. Each well (Costar, Cambridge, MA, USA) was filled with 100 l aliquots of the diluted culture. The plates were incubated at 37 ◦ C for 24 h without shaking, and washed three times with 0.9% (w/v) NaCl. Biofilms were stained with 150 l of 0.1% (w/v) crystal violet solution for 30 min, washed twice with 0.9% (w/v) NaCl and incubated in 200 l of 30% (v/v) acetic acid for 15 min to extract the crystal violet retained by the cells. The extract was used to determine the amount of biofilm by measuring its A590 with a microtiter plate reader (DynaTech, Chantilly, Va.). For inhibition studies, TSB medium containing DspB at different concentrations was used to dilute the overnight culture of bacteria. For detachment studies, biofilms were formed using non-DspB TSB medium, and then treated by DspB at different concentrations for 1 h. At least six replicates were conducted for each sample, and each experiment was performed at least in triplicate.
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0.30 mg/ml. When the enzyme concentration was high enough, the nanoparticles were almost saturated due to the fixed amount of enzymes immobilized on the surface of the nanoparticles (Gan et al. 2012). 3.2. Effect of temperature and pH on activity of free and immobilized DspB The activity of the free and immobilized DspB increased up to 40 ◦ C and then decreased with further increases in temperature. The optimum temperature for the free and immobilized DspB was 40 ◦ C (Fig. 2 A). And the optimum pH was pH 6.0 for both enzymatic forms (Fig. 2B). Therefore, immobilization of DspB on CMCS nanoparticle does not change its optimum temperature and pH. Fig. 1. Transmission electron micrograph of nanoparticles. The samples were immobilized on copper grids and dried at room temperature. Then they were stained with phosphate tungsten acid and examined by TEM.
3. Results and discussion 3.1. Determination of DspB loading capacity and efficiency The size and morphology of the nanoparticles were observed by transmission electron microscopy (TEM) (Fig. 1). TEM images showed that nanoparticles have nearly spherical shape and smooth surface. And the size range of the nanoparticles was between 100 and 500 nm. The loading capacity and efficiency of DspB were affected by its concentration (Table 1). As the concentration of DspB increased in the range 0.05–0.40 mg/ml, the loading efficiency decreased from 79.25 ± 1.50% to 39.52 ± 0.08%. On the other hand, the DspB loading capacity was enhanced dramatically from 198.13 ± 3.75 mg/g to 767.08 ± 13.90 mg/g by increasing the DspB concentration from 0.05 to 0.30 mg/ml. However, the loading capacity did not increase significantly from 0.30 to 0.40 mg/ml, indicating that nanoparticles were almost saturated by DspB at
3.3. Stability of free and immobilized DspB 3.3.1. Thermal stability of free and immobilized DspB The effect of temperature on the stability of free and immobilized DspB at pH 6.0 was shown in Fig. 3. The activities of both free and immobilized DspB were not adversely affected at temperatures below 40 ◦ C. The residual activity dropped significantly above 40 ◦ C. However, compared with free enzyme, immobilized DspB lost less activity and retained 21.0% of its initial activity after incubation at 70 ◦ C for 60 min, whereas the free DspB lost almost all activity. The improved thermal stability for the nanoparticle-coupled enzyme is likely because some enzyme molecules have been entrapped into the core of nanoparticles, which could provide more rigid external backbone for enzyme molecules and protect
Table 1 DspB loading capacity (LC) and loading efficiency (LE). DspB (mg)
LE (%)
0.05 0.10 0.20 0.30 0.40
79.25 66.42 59.52 51.14 39.52
LC (mg/g) ± ± ± ± ±
1.50 0.07 1.84 0.93 0.08
198.13 332.08 595.21 767.08 790.42
± ± ± ± ±
3.75 0.36 18.44 13.90 1.56
Fig. 3. Thermal stability of free (open triangles) and immobilized (closed triangles) DspB. The enzyme was incubated in 50 mM phosphate buffer (pH 6.0) without substrate for 1 h at 0–70 ◦ C, respectively. Residual activities were measured at 40 ◦ C and pH 6.0. The activity of enzyme without incubation was assigned a value of 100%.
Fig. 2. Effect of temperature (A) and pH (B) on the activity of free and immobilized DspB. (A) The optimal temperature of free (open triangles) and immobilized (closed triangles) DspB was determined by measuring the activity in 50 mM sodium phosphate buffer (pH 6.0) at various temperatures (20–80 ◦ C). (B) The optimal pH of free (open triangles and circles) and immobilized (closed triangles and circles) DspB was determined by measuring the activity at 40 ◦ C in 50 mM citrate acid buffer (pH 4.0–5.6; triangles), phosphate buffer (pH 5.6–8.0; circles). The enzymatic activity of a fresh sample of free or immobilized DspB measured at pH 6.0 and 40 ◦ C was defined as 100%.
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Fig. 4. Storage stability of free (open triangles) and immobilized (closed triangles) DspB. The enzyme was incubated in 50 mM phosphate buffer (pH 6.0) without substrate at 37 ◦ C for different periods of time, respectively. Residual activities were measured at 40 ◦ C and pH 6.0. The activity of enzyme without incubation was assigned a value of 100%.
the enzymatic configuration from distortion or damage by heat exchange (Nawani et al. 2006). 3.3.2. Storage stability of free and immobilized DspB Fig. 4 showed the storage stability of the free and immobilized DspB at 37 ◦ C. The free DspB was stable for short incubation periods but was adversely affected at longer incubation time, losing its activity completely within 7 h at 37 ◦ C. In contrast, the immobilized enzyme retained 33.4% of its initial activity after 7 h. These results indicated that enzyme storage stability was significantly improved by immobilization. The increased stability of immobilized enzyme may result from multiple interactions (e.g. electristic interaction, hydrogen bonding and hydrophobic interaction) between DspB and the nanoparticles (Ren et al. 2011). Enzymes are quite sensitive against the environmental situation and may lose their activities easily, thus, the stability of immobilized enzyme is extremely important for subsequent applications, especially in vivo. 3.3.3. Reusability of immobilized DspB The reusability of DspB immobilized on CMCS nanoparticles has also been studied because of its importance for industry to reduce the processing costs. After 8 cycles, the activity of immobilized DspB maintained at a level exceeding 60% of the original activity (Fig. 5). The loss of immobilized DspB activity may be attributed to the conformational changes during reuse or the leakage from the support matrix (Keerti Gupta et al., 2014). From a commercial point of view, the reusability of enzymes is one of the most important features for the immobilization. The result showed that the immobilized enzyme had excellent operational stability. Such reusability could significantly reduce the
Fig. 5. Reusability of immobilized DspB. The used enzyme was washed with distilled water, then added to a substrate solution to start a new cycle. Activity found for each repetition was compared with the initial activity, assuming it was 100%.
operation costs in applications. Moreover, immobilized DspB on nanoparticles can be used on the surface of medical materials in vivo to prevent biofilm formation, which can greatly extend the range of application of this enzyme. The results of immobilization strongly depend on the properties of supports (Wang et al. 2009). Recently, nanotechnology has shown a significant promise in the preparation of immobilized enzymes. Nanomaterials embody distinctive physicochemical and biological properties, such as small size, magnetism and large surface area to volume ratio, which make them favorable for enzyme immobilization (Li et al. 2008; Rai et al. 2009). Immobilization of enzymes on nanoparticles can improve their resistance to denaturing compounds, stability to pH and temperature, as well as an adequate environment for their repeated use (Fang et al. 2009). In addition, the irreversible aggregation of enzyme on heating can be completely prevented by complexation between the heatdenatured enzyme and nanoparticles according to a mechanism similar to that of a molecular chaperone (Takehiro et al., 1994; Akiyoshi et al., 1999; Nomura et al., 2003). Chitosan is an attractive natural biopolymer with large primary amine groups and positive charge. It has shown excellent biocompatibility, susceptibility to chemical modifications, as well as the ability to increase membrane permeability (Colonna et al. 2008; Yang et al. 2010). Moreover, chitosan nanoparticle can easily be prepared, which is one of the most promising support material for enzyme immobilization (Wang et al. 2013; Zang et al. 2014; Long et al. 2015). It is reported that the performance of -galactosidase immobilized on chitosan nanoparticles was higher than that immobilized on chitosan micro- and macroparticles (Biro et al. 2008). This can be ascribed to the better distribution of the enzyme on the nanoparticles due to high specific surface area of chitosan nanoparticles and large of active functional groups available for fixing the enzyme molecules (Tang et al. 2006).
3.4. Antibiofilm activity of free and immobilized DspB 3.4.1. Inhibition of biofilm formation by free and immobilized DspB Biofilm formation of S. epidermidis RP62A was significantly inhibited by both free and immobilized enzymes in a concentration-dependent manner (Fig. 6). Immobilized DspB showed greater antibiofilm activity than free enzyme. When the concentration of DspB is 0.30 U/ml, 81.5% of biofilm formation of S. epidermidis RP62A was inhibited by free enzymes and 93.8% by immobilized enzyme. To determine the antibiofilm activity spectrum of free and immobilized DspB, the effects of DspB on biofilm formations of various strains (S. aureus, S. epidermidis and A. actinomycetemcomitans) were measured using 96-well microtiter plate assay. The strains were cultured in the presence of 0.30 U/ml DspB for 24 h to form biofilms. As shown in Fig. 7 A, biofilm formations by all of 9 strains were inhibited by both free and immobilized DspB. Immobilized DspB exhibited stronger inhibition effect (>90%) than free DspB (≈80%). Interestingly, CMCS nanoparticles also exhibited prevention effect on biofilm formation (from 6% to 50% at 0.156 to 2.500 mg/ml). In our previous study, CMCS prevented initial bacterial adherence and cell–cell interaction, which is involved in the aggregation through charge neutralization and flocculation by bridging mechanism. CMCS nanoparticles does not affect the cationic charge, but also exhibited a certain antibiofilm activities. Additionally, the immobilized enzyme enhanced stability of DspB, which increased biofilm formation inhibition effect of DspB. These results together may explain the mechanism of the increased biofilm formation inhibition effect of immobilized DspB.
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Fig. 6. Inhibition of S. epidermidis RP62A biofilm formation by nanoparticles, free and immobilized DspB in 96-well microtiter plates. (A) The strains from an overnight culture were diluted to 1 × 106 colony-forming units (CFU)/ml with TSB media containing DspB at different concentrations (range 0–0.3 U/ml). After 24 h cultivation, wells were rinsed and stained with crystal violet. (B) Quantitation of crystal violet staining in panel A. Wells were destained with acetic acid and the absorbance of the crystal violet solution was measured at 590 nm. Absorbance is proportional to biofilm biomass.
Fig. 7. Inhibition (A) and detachment (B) effect of nanoparticles, free and immobilized DspB on biofilm formations in 96-well microtiter plates. For inhibition studies, TSB medium containing 0.3 U/ml DspB was used. For detachment studies, bacteria were cultured in non-DspB TSB medium for 24 h to form mature biofilm, then treated with 0.3 U/ml DspB for 1 h. Wells were stained with crystal violet and destained with acetic acid. The absorbance of the crystal violet solution was measured at 590 nm. Absorbance is proportional to biofilm biomass.
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3.4.2. Detachment of mature biofilm by free and immobilized DspB The detachment effect of DspB on mature biofilms was also explored. The strains were cultured for 24 h to form mature biofilms, and then biofilms were treated by DspB for 1 h. As shown in Fig. 7B, both free and immobilized DspB disrupted the preexisting biofilms of all 9 strains. Immobilized DspB showed similar effect with free DspB (≈80%), which is ascribed to the comparable activity of free and immobilized enzymes. 4. Conclusion In this work, DspB was loaded on nanoparticles of LA-CMCS. The immobilization enhanced thermal stability, storage stability and reusability of DspB significantly. The immobilized enzyme was more effective than free enzyme in inhibiting and detaching the biofilm formations by several bacterial species such as S. aureus, S. epidermidis and A. actinomycetemcomitans. The studies also show that CMCS nanoparticles can exhibit prevention effect on biofilm formation, which can be used as a kind of excellent delivery of antibiofilm agents. These results suggest that DspB immobilized on CMCS nanoparticles can be used for treatment of medical device-associated infections. Moreover, the complex of DspB and nanoparticles can eventually be expanded in vivo, which will further broaden its range of application. Acknowledgements This work was supported by National High-tech R&D Program (2014AA093504 and 2007AA091506), National Natural Science Foundation of China (U1406402), Science and Technology Development Project of Shandong Province (2014GGH215002). References Akiyoshi K, Sasaki Y, Sunamoto J. Molecular chaperone-like activity of hydrogel nanoparticles of hydrophobized pullulan: thermal stabilization with refolding of carbonic anhydrase B. Bioconjugate Chem 1999;10(3): 321–4. Alves D, Pereira MO. Mini-review: antimicrobial peptides and enzymes as promising candidates to functionalize biomaterial surfaces. Biofouling 2014;30(4): 483–99. Augustine N, Kumar P, Thomas S. Inhibition of Vibrio cholerae biofilm by AiiA enzyme produced from Bacillus spp. Arch Microbiol 2010;192(12): 1019–22. Badel S, Laroche C, Gardarin C, Bernardi T, Michaud P. New method showing the influence of matrix components in Leuconostoc mesenteroides biofilm formation. Appl Biochem Biotech 2008;151(2-3):364–70. Biro E, Nemeth AS, Sisak C, Feczko T, Gyenis J. Preparation of chitosan particles suitable for enzyme immobilization. J Biochem Biophys Methods 2008;70(6):1240–6. Brindle ER, Miller DA, Stewart PS. Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB. Biotechnol Bioeng 2011;108(12):2968–77. Chauhan N, Narang J, Sunny Pundir CS. Immobilization of lysine oxidase on a goldplatinum nanoparticles modified Au electrode for detection of lysine. Enzyme Microb Technol 2013;52(4-5):265–71. Colonna C, Conti B, Perugini P, Pavanetto F, Modena T, Dorati R, et al. Ex vivo evaluation of prolidase loaded chitosan nanoparticles for the enzyme replacement therapy. Eur J Pharmaceutics Biopharmaceutics 2008;70(1): 58–65. Elchinger PH, Delattre C, Faure S, Roy O, Badel S, Bernardi T, et al. Effect of proteases against biofilms of Staphylococcus aureus and Staphylococcus epidermidis. Lett Appl Microbiol 2014;59(5):507–13. Fang H, Huang J, Ding L, Li M, Chen Z. Preparation of magnetic chitosan nanoparticles and immobilization of laccase. J Wuhan Univ Technol Mater Sci Ed 2009;24(1):42–7. Fine DH, Furgang D, Kaplan J, Charlesworth J, Figurski DH. Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite. Arch Oral Biol 1999;44(12):1063–76. Gan Z, Zhang T, Liu Y, Wu D. Temperature-triggered enzyme immobilization and release based on cross-linked gelatin nanoparticles. PloS One 2012;7(10):e47154.
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