Development and Characterization of a Novel Bioactive Polymer withAntibacterial and Lysozyme-Like Activity

Maria Rachele Guascito,1 Daniela Chirizzi,2 Cosimino Malitesta,1 Livia Giotta,1

Disma Mastrogiacomo,1 Ludovico Valli,1 Loredana Stabili1,2,3

1 Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universit�a del Salento, Via Monteroni 73100, Lecce, Italy

2 Dipartimento di Beni Culturali, Universit�a del Salento, Via Birago 73100, Lecce, Italy

3 Istituto per l’Ambiente Marino Costiero CNR U.O.S. Taranto, Via Roma 3 74100, Taranto, Italy

Received 5 March 2013; revised 31 July 2013; accepted 27 August 2013

Published online 2 September 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22404

ABSTRACT:

The development and characterization of a novel bioac-

tive polymer based on the immobilization of glucose oxi-

dase enzyme (GOx) in a polyvinyl alcohol (PVA) film

showing antibacterial activity is presented. The PVA-

GOx composite material was extensively characterized by

UV-vis, X-ray Photoelectron (XPS) spectroscopy and by

Fourier Transform Infrared (FTIR) spectroscopy to verify

the preservation of enzyme structural integrity and activ-

ity. The antimicrobial activity of this composite material

against Escherichia coli and Vibrio alginolyticus was

assessed. Furthermore the lysozyme-like activity of PVA-

GOx was highlighted by a standard assay on Petri dishes

employing Micrococcus lysodeikticus cell walls. The find-

ings from this study have implications for future investi-

gations related to the employment of PVA-GOx system as

a composite material of pharmaceutical and technologi-

cal interest. VC 2013 Wiley Periodicals, Inc. Biopolymers

101: 461–470, 2014.

Keywords: antibacterial; glucose oxidase; lysozyme-like

activity; polyvinyl alcohol; XPS; FTIR

This article was originally published online as an accepted pre-

print. The “Published Online” date corresponds to the preprint

version. You can request a copy of the preprint by emailing the

Biopolymers editorial office at biopolymers@wiley.com

INTRODUCTION

Microbial infection is one of the most serious con-

cerns for many applications such as biotechnol-

ogy, pharmaceutical, textiles, food packaging

and storage, shoe industry, water purification,

medical devices, and dental surgery equip-

ment.1,2 Nevertheless the development of efficient devices

based on biocide surfaces is a major challenge for modern

scientists.3 Most antibacterial systems developed to date

show some toxicity and short-term antimicrobial action.4–7

Moreover, in an attempt to control bacteria, prophylactic use

of antibiotics has become a frequently used strategy in

human therapy, veterinary and food production. Antibiotics,

which have been used in large quantities, are in many cases

ineffective, or result in increases in virulence due to the

development of bacterial resistance.8

As a consequence, new antimicrobial agents have recently

gained considerable interest from both an industrial and

research point of view because of their potential to provide

safety benefits to a wide range of materials. Nowadays scientists

need indeed to design materials with a surface that has a very

broad spectrum of biocide activity, killing via a mechanism

which does not result in the emergence of resistant strains, and

that can be used repeatedly, thus resulting appropriate for

development of biomedical devices.9,10 These materials must

show however chemical, physical and thermo-mechanical

properties (porosity, flexibility, elasticity, corrosion resistance)

making them suitable as support for antimicrobial agents.11–13

Correspondence to: Maria Rachele Guascito; e-mail: maria.rachele.guascito@

unisalento.it

Contract grant sponsor: Fondazione Cassa di Risparmio di Puglia (Viale della

Repubblica 111, Bari–Italy)

VC 2013 Wiley Periodicals, Inc.

Biopolymers Volume 101 / Number 5 461

The most noteworthy antibacterial substance in living

organisms is lysozyme. This enzyme is present in mucus and

secretions and is the best characterized antimicrobial peptide

(AMP) involved in the defence against bacteria.14 The lyso-

zymes dissolve Gram-positive bacteria by breaking the b-

glycosidic bond between C1 of N-acetylmuramic acid (NAM)

and C4 of N-acetylglucosamine (NAG), which are the saccha-

ride components of the peptidoglycan layer in bacterial cell

wall, thus behaving as a glycosidase. A number of additional

antimicrobial enzymes are widespread in nature, where they

play a critical role in defending living organisms from bacterial

attack. Three major classes of enzymes acting against bacteria

can be acknowledged, i.e., proteolytic enzymes, polysaccharide

degrading enzymes, and oxidative enzymes.15 New materials

able to mimic the action mechanism of these biological systems

represent an interesting biotechnological innovation for a num-

ber of application fields, mainly because they are expected to

exhibit high biocompatibility. In this context several investiga-

tions aimed to develop antibacterial agents by immobilization

of antimicrobial enzymes into polymer matrices have been

reported.16 As an example lysozyme has been immobilized in

different polymer substrates such as PVA beads, nylon 6,6 pel-

lets, cellulose triacetate films, and chitosan films 17. Moreover

the biocide efficacy of a lysozyme-PVA composite material has

been tested by Conte et al.19 via an optical assay for the lysis of

Micrococcus lysodeikticus cell walls, a well-established test for

monitoring lysozyme enzymatic activity. Amitai et al.20,21 have

recently proposed the use of multifunctional polymers based

on enzyme-polymer matrices for the generation of perishable

substances (free halogens) as highly active and rapid disinfec-

tants. In particular they have proposed a new synthetic antimi-

crobial material based on the incorporation of leukocyte

mimicking enzymes (horse radish peroxidase and glucose oxi-

dase) into a polyurethane film acting in presence of benign

renewable resources such as water, sugar, and salt.

In this work we describe the development and characteriza-

tion of a novel antibacterial material based on GOx immobi-

lized in a PVA film. PVA represents an ideal enzyme

immobilization material due to the abundance of hydroxyl

groups providing a microenvironment similar to the enzyme’s

natural environment.22,23 It has been in fact widely used

because of its inherent good biocompatibility and desirable

physical properties, such as elastic nature and good film-

forming properties.24 GOx acts in presence of glucose to gener-

ate by-products as oxygen species (e.g., H2O2,.O2-,.OH) that

are endogenous and exogenous toxic products for microbes in

vivo.25 Despite to the well-known antimicrobial activity of

GOx26 and the valuable properties of PVA in stabilizing this

enzyme,27 the application of a GOx-PVA composite system as

a biocide material was limited to the field of food preserva-

tion.28 The antibacterial packaging material developed by Ge

et al.28 contained however supplementary antimicrobial agents

such as chitosan and tea extract. Nevertheless the immobiliza-

tion of GOx into PVA matrices was exploited in several bio-

sensing applications, using different methodologies for

preparing the films, included the recently come back into

favour electrospinning techniques.29–31

The PVA-GOx composite material described in this work

contains only GOx as antimicrobial agent and is prepared by a

simple procedure. The bio-polymer has been fully characterized

by different spectroscopic techniques and its ability to inhibit

bacterial growth in presence of glucose has been investigated.

The antibacterial lysozyme-like activity has been further eval-

uated by a standard assay on Petri dishes employing Micrococcus

lysodeikticus dried cell walls. The results achieved, highlighting

the antimicrobial properties of enzyme-polymer matrices based

on GOx and PVA, are particularly interesting since these systems

represent one of the most important typology of materials used

in biosensors for glucose in blood self-testing.32 In this context

the investigation of biocidal properties of GOx-based active

layers employed in glucose biosensors is very useful in order to

assess the self-disinfecting potential of these systems and conse-

quently their applicability in continuous real-time glucose moni-

toring devices inserted directly into the blood stream or

subcutaneously. Likely infections occurring at the implantation

site upon insertion of biomedical probes in living tissues repre-

sent indeed one of the major limitations of intravascular and

subcutaneous implantable glucose sensing systems.33,34

EXPERIMENTAL

Materials

Glucose Oxidase (type VII from Aspergillus niger; 174,000 units/

g), b-D-glucose and hydrogen peroxide 30%, were obtained

from Sigma. Na2HPO4, NaH2PO4, poly(vinyl alcohol) film

(PVA, product number Z300381) were purchased from Aldrich.

Micrococcus lysodeikticus cells were obtained from Sigma. All

reagents were analytical grade. Ultrapure water (Millipore Milli-

Q, 18.2 MXcm-1) was used for preparation of all aqueous solu-

tions. 1 M stock glucose solutions (prepared every 2 weeks in a

phosphate buffer solution (PB) pH 5 7.0, I 5 0.2 were allowed

to mutarotate at room temperature overnight before use.

METHODS

Preparation of Antibacterial Composite MaterialThe PVA-GOx composite material was prepared according to the pro-

cedure reported in literature.23 Briefly 3mg of GOx were dissolved in

1 mL aqueous solution containing 10% PVA in ultrasonic bath for 5

462 Guascito et al.

Biopolymers

min. The mixture was kept at room temperature for 6 h and then

stored at 218�C for 48 h.

X-Ray Photoelectron Spectroscopy (XPS)First, Pt foil surface was mechanically polished with alumina powder

and ultrapure water. After pre-treatment procedure composite material

was prepared by drop casting 100 lL PVA-GOx mixture onto Pt foils

(area 3 cm2) leaving it to evaporate for 1 h at room temperature. The

same method was used for plain PVA sample and, for comparison, XPS

spectra of GOx powder were also collected. XPS analysis was carried

out using a Leybold LHS10 spectrometer equipped with an unmono-

chromatized AlKa source (operating at 10 kV and 17 mA) and a SPECS

multi-channel detector. Survey (WS) spectra were recorded in fixed

retarding ratio (FRR) mode with a retarding ratio of B530 and energy

step of 1.0 eV, while high-resolution (HR) regions were acquired in

fixed analyzer transmission (FAT) mode at a pass energy of E0 5 30 eV

with energy step of 0.1 eV. HR spectra relevant to C1s N1s, O1s, and

valence band (VB) signals were recorded. Surface charging was calcu-

lated considering C1s (BE5 285.0 eV) as references35 in order to esti-

mate correct Binding Energy (BE) for each signal. The data analysis and

peak deconvolution were performed by means of a suitable software

New Googly36 which allows satellites and background correction as well

as curve-fitting of photoelectronic peaks.

Fourier Transform Infrared spectroscopy (FTIR)Mid-infrared spectra were acquired with a PerkinElmer Spectrum One

FTIR spectrometer equipped with a Deuterated Triglycine Sulfate (DTGS)

detector. The spectral resolution used for all experiments was 4 cm21. Typ-

ically 16 interferograms were acquired and averaged for each spectrum.

Spectra of dried glucose oxidase, Micrococcus lysodeikticus cells and

D-glucose were recorded by means of an Attenuated Total Reflection

(ATR) accessory. In this case the Internal Reflection Element (IRE)

was a three bounce 4 mm diameter diamond microprism (DuraSam-

plIR, Smiths Detection). A good adhesion between sample and IRE

was achieved by preparing films directly onto the diamond prism by

casting/evaporation technique. For this purpose 3–5 mL of a fresh pre-

pared aqueous solution were deposited on the upper face of the ATR

crystal and the solvent was allowed to evaporate completely. The back-

ground spectrum was acquired using the bare ATR crystal.

A variable specular reflection device designed specifically for thin

film analyses (AmplifIR, Smiths Detection) was instead employed for IR

measurements on PVA films deposited on platinum slides. In this device

the IR beam is externally reflected between the sample surface and an

adjustable gold coated mirror. According to transflectance principles the

IR beam passes through the film, interacts with the sample and is

reflected by the platinum plate towards the gold mirror. The number of

specular reflections achieved before the beam is delivered to the detector

is controlled by adjusting the distance of the mirror to respect the sam-

ple, thus allowing to set the effective pathlength suitable for attaining an

appropriate measurement sensitivity. In this case the background spec-

trum was acquired placing in the device a neat gold slide.

Ultraviolet-Visible Spectroscopy (UV-vis)UV-vis absorption measurements were carried out by means of a

Cary 50 spectro-photometer (Varian). GOx aqueous solutions (500

units/mL) both in 10% PVA and in PB (pH 5 7.0, I 5 0.2) were ana-

lysed using a 0.5 cm optical path Hellma quartz cuvette. For acquisi-

tion of transmission UV-vis spectra of dry PVA-GOx films, 100 mL of

the same solutions were spread onto the external face of an Hellma

quartz cuvette and the solvent was allowed to evaporate for 1 h at

room temperature. Then, the film-coated cuvette was suitably placed

into the sample holder of the spectrophotometer in order to achieve

the optimal interaction between the incident beam and the cast film.

Microbial StrainsFor running the antibacterial assay one human (Escherichia coli) and

one animal marine pathogenic (Vibrio alginolyticus) strain previously

isolated 37 were used. Strains were routinely maintained at 14�C on

Luria–Bertani (LB) Nutrient Agar or Marine Agar 2216 (Beckton

Dickinson and Company) used for the human pathogenic strain and

the marine strain respectively. Bacterial strains were always harvested

during the exponential growth phase, i.e. after 4 h culture at 37�C for

E. coli, or 24�C for V. alginolyticus under constant rotation at 100 rpm.

Antimicrobial ActivityTo test antimicrobial activity, 50 mL of PVA-GOx mixture supplemented

with glucose (10 mM) were added to 10 mL of bacterial suspension (108

cells/mL). Normal growth controls consisted of bacteria incubated with

sterile liquid broth. After 30 min at room temperature, with gentle stir-

ring at 100 rpm, serial dilutions of these suspensions were incubated in

Petri dishes between two layers of the above described specific nutritive

agar to obtain imprisoned bacterial forming punctiform colonies [tech-

nique of "colony forming units" (CFU)]. Routinely, 1023, 1024, and

1025 dilutions were plated out in triplicate. Percentages of inhibition

were determined from the differences in colony numbers in controls

and tests, after 24-h incubation at 24�C for marine strain and at 37�Cfor the pathogenic strain. The percentage of bacteria inhibited by PVA-

GOx in presence of glucose was inferred from the difference between

the number of emerging colonies in controls and in tests.

Lysozyme-Like Activity EvaluationTo detect lysozyme-like activity, inoculated Petri dishes were used as

standard assay.38 Briefly, 700 lL of 5 mg/mL of dried Micrococcus lyso-

deikticus cells were diluted in 7 mL of 0.05M PB-agarose (1.2%)

(pH 5 6.0), then spread on a Petri dish. When the agarose gel has solidi-

fied, 6.3 mm diameter wells were sunk and filled with 30 lL of sample.

The diameter of the cleared zone of the four replicates was

recorded after overnight incubation at 37�C. The positive control was

represented by hen-egg-white lysozyme (Merck, Darmstadt, Ger-

many) at a concentration ranging from 0.5 mg/mL to 1.5 mg/mL and

producing diameters of lysis comprised between 3.5 and 10.5 mm.

The effects of pH, ionic strength (I), and temperature were examined.

The pH effect was tested by dialyzing the samples in PB 0.05M, ionic

strength, I 5 0.175, adjusted at pH 4, 5, 6, 7, 8, and by dissolving

agarose in PB at the same I and pH values. The ionic strength effect

was tested in PB 0.05 M (pH 5 6.0), adjusted at I 5 0.0175, 0.175,

1.75, and agarose was dissolved in PB at the same I values. The tem-

perature effect was tested with incubations of samples (in PB,

pH 5 6.0, I 5 0.175) at 5, 15, 22, and 37�C.

RESULTS AND DISCUSSION

X-Ray Photoelectron SpectroscopyXPS was used to analyze the surface chemistry of PVA-GOx

composite film. Comparison between WS spectra in Figure 1

Development and Characterization of a Novel Bioactive Polymer 463

Biopolymers

of PVA pristine film (curve a), PVA-GOx (curve b), and GOx

powder controls (curve c) shows, as expected, a surface with

intense C1s and O1s signals and respective their Auger one.

Moreover the presence of N (from enzyme structure) in

PVA-GOx composite material and in GOx standard powder

was confirmed, while the N1s signal was absent in PVA film. In

fact, detailed spectra in the N1s region showed, in the PVA-

GOx film and GOx powder, the presence of component peaks

characteristic of the GOx standard. Detailed spectra of Pt4f

region showed the absence of the signal relative to Pt4f on each

sample. Finally, also the VB region for these samples is repre-

sentative of PVA composition.39,40 These collected data con-

firm that the composite material is compact and uniformly

distributed on the Pt surface.23

The typical surface elemental composition (see Table I) and

bonding with respect to C1s, O1s, N1s core level spectra were

determined at film surface (depth of �5 nm). Both PVA pris-

tine film and GOx powder exhibit in terms of peaks position

and composition % values according to previous reported

data.41,42 The complete PVA-GOx composite material was

composed of �2.8% N confirming the presence of the enzyme

with a relatively high degree of GOx immobilization. For com-

parison GOx powder shows a value of �4.5% N. XPS was also

used to determine O/C and N/C ratios to compare protein and

PVA fingerprints with PVA-GOx system. Analysis of GOx and

PVA standard showed respectively a O/C ratio of �0.35 and

�0.48 (theoretical value 0.5). As a consequence for PVA-GOx

materials the O/C ratio of 0.50 is very close to the PVA not

modified film. Moreover the N/C ratio may be a more appro-

priate measure of the interaction of the enzyme chemistry at

the surface and for samples PVA-GOx the N/C ratio of 0.04 is

quite near 0.06 value recorded for GOx control, confirming the

enrichment of the PVA surface in GOx enzyme.

A deeper study on C1s and N1s HR regions was also

performed. In Figure 2 detail of high-resolution spectra of C1s

(a, b, and c) and N1s (a’, b’, and c’) is shown. The respective

carbon species are marked by an asterisk. The C1s spectrum of

PVA has two main peaks corresponding to the aliphatic carbon

(C*-C) and the oxydrilic carbon (C*-OH) respectively at

BE 5 285.0 6 0.1 eV and BE 5 286.5 6 0.1 eV.43 A third com-

ponent at BE 5 289.0 6 0.1 eV was also observed and attrib-

uted to the carboxylic carbon group (C*OO-). The C1s

spectrum of GOx powder has been fitted also by using three

peak components and corresponding respectively to the char-

acteristic aliphatic carbon signal (C*-C) at 285.0 6 0.1 eV, that

may have originated also from surface contaminations, to the

amidic and aminic carbon at 287.3 6 0.1 eV (both indicate

C*-N), as expected for peptide chains, and finally to the car-

boxylic carbon group (C*OO-) at 288.9 6 0.1 eV. For both

standard the carboxylic carbon group functionality has been

confirmed in IR analysis.

The C1s spectrum of PVA-GOx composite film shows,

instead, four peak components. Any attempt to fit the experi-

mental data by using only three components has failed, even if

a wider Full Width (FW) was used as fitting parameter. It was

necessary to add a new component as reported in Figure 1c.

The aliphatic carbon component (C*-C) is at 285.0 6 0.1 eV

and the alcoholic carbon component (C*-OH) at 286.1 6 0.1

eV, moreover the third peak (C*-N) at 287.5 6 0.1 eV is the

characteristic of the amidic and aminic carbon in peptide

chains. Finally an over-oxidized component (C*ox) is evident

at 289.6 6 0.1 eV. These results confirm that the PVA-GOx

material is a composite hybrid film according to the presence

of N1s signal. In fact the HR N1s spectrum of PVA (Figure 2a’)

shows only a negligible trace, while the N1s spectra of GOx

powder and PVA-GOx composite film (Figures 2b’ and 2c’)

show both the peak at BE 5 400.1 6 0.1 eV.

Fourier Transform Infrared SpectroscopyFigure 3 presents the ATR-FTIR spectra of dry glucose oxidase

and Micrococcus lysodeikticus dried cells deposited on the dia-

mond microprism by casting/evaporation. Both samples con-

tain peptide and saccharide moieties, thus producing similar

spectroscopic patterns. Saccharides account mainly for broad

bands between 1000 and 1100 cm21 (alcoholic C-O stretching

modes), whilst peptide chains and acetamide moieties (C5O,

N-H bonds) strongly contribute to signals centered at �1640

and �1540 cm21 (Amide I and Amide II bands respectively).

FIGURE 1 WS spectra of PVA (curve a), GOx-PVA (curve b) and,

for comparison, of GOx standard powder (curve c) deposited onto

Pt foils.

Table I XPS Elemental Quantitative Results

Sample C1s[%] O1s[%] N1s[%] O/C N/C

PVA 67.32 32.54 traces 0.48 —

GOx powder 70.57 24.89 4.54 0.35 0.06

PVA-GOx 64.58 32.62 2.81 0.51 0.04

Values shown are as a percentage of the total.

464 Guascito et al.

Biopolymers

A band at 1396 cm21 is particularly intense in Micrococcus

lysodeikticus spectrum. According to recent investigations on

bacterial layers by FTIR difference spectroscopy,43 this band

can be mainly assigned to the symmetric COO- stretching

mode of free glutamate groups, particularly abundant in the

cell wall peptidoglycan layer. These spectra were used for iden-

tifying marker bands of glucose oxidase and Micrococcus lyso-

deikticus cells in composite PVA films.

Infrared characterization of PVA films was achieved on the

same support (platinum foil) employed for XPS

FIGURE 2 C1s and N1s detailed spectra for PVA film (a, a’), GOx powder (b, b’) and PVA–GOx

composite film (c, c’).

Development and Characterization of a Novel Bioactive Polymer 465

Biopolymers

measurements. Due to the high sensibility of the multireflec-

tion technique adopted, a very thin layer of PVA and biological

components was deposited by soaking the Pt slides in diluted

solutions (the dilution factor was 10 for each solution

employed in comparison to the original ones) and allowing the

solvent to evaporate completely.

The infrared spectrum of pure PVA film deposited in this

way on the platinum slide is presented in Figure 4 (trace A).

PVA is a semicrystalline synthetic polymer able to form some

domains, affecting the spectroscopic features, depending on

several process parameters44 and its basic properties depend on

the degree of polymerization and on the degree of hydrolysis.

Residual acetate moieties in our PVA samples are revealed by

the signals at 1737 cm21 (ester C@O stretching), 1375 cm21

(-CH3 bending), and 1246 cm21 (ester C-O stretching).

Moreover, in agreement with the polyalcohol structure, the

spectrum shows a wide and intense band due to the presence

of hydrogen bonded hydroxyl groups (O-H stretching) cen-

tered at 3377 cm21, a band at 2943 cm21 corresponding to the

-CH2- stretching vibration, and a broad signal of medium

intensity at 1436 cm21, which can be attributed mainly to

-CH2- and -CH3 deformation modes. Alcoholic C-O bonds

account for the absorption peak at 1097 cm21 and surround-

ing bands. PVA composite films containing glucose oxidase

(trace B) shows the amide I (1655 cm21) and amide II (1555

cm21) bands of proteins overlapped to the above mentioned

features of the polymer. The intensity of marker bands for pro-

tein samples further increase when Micrococcus lysodeikticus

cells are added to the PVA-GOx mixture employed for prepar-

ing the film (trace C). The trace D represents the infrared spec-

trum recorded on this film after soaking the slide in a 10 mM

D-glucose solution. The spectrum is dominated by a strong

absorption band at 1737 cm21, which indicates the formation

of an ester upon contact of the film components with glucose.

To demonstrate that this compound is generated by the

GOx-catalized reaction of D-glucose with oxygen, 1 mL of a

pH 7 buffered solution containing glucose (32 mg/mL) and

potassium phosphate (10 mM) was supplemented in different

tubes with the following components: glucose oxidase (final

concentration 80 mg/mL), both Micrococcus lysodeikticus cells

and GOx (final concentration 100 mg/mL and 80 mg/mL,

respectively), only Micrococcus lysodeikticus cells (final concen-

tration 100 mg/mL). The aerobic conditions adopted guarantee

the presence of dissolved oxygen in all solutions. The tubes

were vortexed for 30 s and 3 mL aliquots of each sample were

deposited on the ATR prism of the infrared spectrometer.

FIGURE 3 ATR-FTIR spectra of dry films of Micrococcus lysodeik-

ticus cells (trace A) and glucose oxidase from Aspergillus niger

(trace B).

FIGURE 4 Multiple specular reflectance FTIR spectra of thin

films deposited on platinum slides by casting/evaporation: plain

PVA film (A), PVA-GOx composite film (B), PVA-GOx-Micrococcus

lysodeikticus cells composite film (C), PVA-GOx-Micrococcus lyso-

deikticus composite film soaked in a glucose solution and subse-

quently dried (D).

466 Guascito et al.

Biopolymers

The resulting ATR-FTIR spectra, acquired after solvent

evaporation, are presented in Figure 5. It is clear that glucose

oxidase addition is responsible for the appearing of a charac-

teristic peak at 1737 cm21, since D-glucose does not show a

similar feature (trace A), even in presence of Micrococcus lyso-

deikticus cells (trace D). On the other hand GOx-induced spec-

tral changes, clearly detectable in trace B are further enhanced

in presence of Micrococcus lysodeikticus cells (trace C), indicat-

ing a role played by the cellular system in the modulation of

the enzyme-catalyzed reaction. In trace C arrows point out

some clear spectral changes, such as disappearing of bands at

1256 cm21, 897 cm21, and 844 cm21 accompanied by the

simultaneous appearing of signals at 1228 cm21 and 874

cm21. These absorption frequency changes likely arise from

modification of the glucose structure, following oxidation of

the aldehyde carbon. However the most informative signal is

the band at 1737 cm21, typical of ester compounds, which

allows concluding that the expected product of the catalytic

reaction (d-gluconolactone) is formed. Moreover the shoulder

at 1770 cm21 reveals that some c-gluconolactone is also

released in the reaction mixture. C5O stretching vibration of

c-lactones are indeed expected to absorb at higher frequencies

due to increased angle strain.45

Even though the wavelength-dependence of the optical path

in ATR modality should be taken into account when compar-

ing transflectance and attenuated total reflectance spectra, it is

possible to compare trace C in Figure 5 and trace D in Figure 4

for getting qualitative information on the chemical composi-

tion of PVA-GOx-Micrococcus lysodeikticus cells composite

films deposited on platinum slide after glucose treatment. It is

clear that features, assigned to d-gluconolactone, dominate the

FIGURE 5 ATR-FTIR spectra of films deposited by casting/evapo-

ration from the following aqueous solutions: glucose 32 mg/ml (A),

glucose 32 mg/ml 1 GOx 80 mg/ml (B), glucose 32 mg/ml 1 GOx 80

mg/ml 1 Micrococcus lysodeikticus cells 100 mg/ml (C), glucose 32

mg/ml 1 Micrococcus lysodeikticus cells 100mg/ml (D).

FIGURE 6 A and B: UV-vis spectra of PVA-GOx aqueous solution

(curve a) and of PVA-GOx film cast on quartz slide (curve b) in dif-

ferent wavelength regions. C: UV-vis spectra of glucose oxidase in

the oxidized (curve c) and fully reduced state (curve d). Full reduc-

tion of the adenine dinucleotide (FAD) prosthetic group was

obtained following addition of glucose 10 mM.

Development and Characterization of a Novel Bioactive Polymer 467

Biopolymers

infrared spectrum of PVA film, almost totally hiding the poly-

mer bands. The signal at 876 cm21 is well defined, indicating

that the glucose/d-gluconolactone conversion is nearly com-

plete in this case.

This result reveals that glucose oxidase is highly active in

the PVA matrix and presumably the association of the polymer

with Micrococcus lysodeikticus cells play a key role in enhancing

the production of d-gluconolactone.

UV-Visible SpectroscopyUV-vis characterization was performed to further verify the

preservation of GOx integrity when it is embedded in PVA

film. For this purpose, GOx spectra were acquired in the range

240–700 nm in PVA-GOx mixture (Figure 6 curve a) and in

GOx-PVA film (Figure 6 curve b).

It is evident that the peak position and the shape of the

absorption band for GOx-PVA film and for PVA-GOx solution

are the same. In fact, Figures 6A and 6B show GOx characteris-

tic peaks at 275, 380, and 453 nm. The UV absorption band at

275 nm is assigned to polypeptide chains in protein structure.

The peaks at 380 and 453 nm are characteristic of the oxidized

form of flavin group.46,47 The similarity of PVA-GOx solution

and PVA-GOx film spectra suggests that PVA entrapment

upon solvent evaporation does not determine any modifica-

tion of enzyme structure in the dry polymer-embedded state.

Moreover, the enzyme activity towards glucose oxidation was

verified by observing on PVA-GOx film (see Figure 6C) the

expected absorption bands modifications after glucose addi-

tion (10 mM) due to enzyme reduction (curve d). The oxi-

dized form of the enzyme (curve c) was easily restored when

solutions or films were saturated with O2.48

FIGURE 7 Antibacterial assay on Petri dishes: Vibrio alginolyticus incubated with PVA/GOx (A)

Vibrio alginolyticus grown in the control (B). Escherichia coli incubated with PVA/GOx (C). Esche-

richia coli grown in the control (D).

468 Guascito et al.

Biopolymers

The above reported results confirm the great biocompatibil-

ity of PVA, already described in the literature.22–24,30 PVA is in

fact considered an ideal enzyme immobilization material since

its water content matches that of biological tissue.

Antibacterial Activity AssessmentOur data enhanced, in vitro, the presence of a conspicuous

antimicrobial activity in PVA-GOx composite material supple-

mented with glucose. This activity, as shown in Figure 7, was

tested on Escherichia coli and Vibrio alginolyticus. By way of

plate assay, we were able to quantify the degree of inhibition

produced by PVA-GOx in presence of glucose on each strain.

Vibrio alginolyticus was the most inhibited (97% growth inhi-

bition) however, Escherichia coli resulted also affected by PVA-

GOx treatment (75% growth inhibition).

Lysozyme-Like Activity AssessmentThe PVA-GOx composite material showed also an antibacterial

lysozyme-like activity. The observed antibacterial activity was

strictly affected by pH and ionic strength (I) of the sample and

of the reaction medium as well as by the incubation tempera-

ture. By the standard assay on Petri dishes the maximum

diameter of lysis (4.20 6 0.02 mm corresponding to 0.6 mg/

mL of hen egg-white lysozyme) was reported at I 5 0.175,

pH5 6.0 and incubation temperature of 37�C. Thus, the lyso-

zyme like activity evidenced in the present study had maxi-

mum of activity at pH and ionic strength values as those

previously reported for other lysozymes.49–51 This activity was

present in all the sampling filled with 30 lL of PVA-GOx in

the presence of glucose 10 mM. By contrast, the wells filled

with PVA-GOx without glucose and the empty ones do not

show lisozyme-like activity (Figure 8).

For further confirmation of the role of GOx and glucose in

the proposed activity, in Figure 7 the results of a second set of

tests prepared with the same procedure for GOx and glucose

were also reported. In this second set the wells were filled with

PVA-GOx and hydrogen peroxide (H2O2) 1.5 mM (test D),

only H2O21.5 mM (test E), and only PVA (test F). As expected,

in these last assays the lysozyme-like activity was not recorded.

The lack of lysozyme-like activity by H2O2 allows to conclude

that the lytic activity of PVA-GOx system in presence of glu-

cose does not arise merely from the final product of the enzy-

matic reaction.

On the basis of these encouraging results, we suggest that

the antibacterial activity arises from chemical interaction

between the enzyme and the sugar, involving likely intermedi-

ate species. The GOx-mediated glucose oxidation triggers the

lysis of bacterial cell walls components, accounting for positive

lysozyme-like activity assay, and leads however to the produc-

tion of an ester (presumably the expected product d-glucono-

lactone), as revealed by infrared spectroscopy.

CONCLUSIONSThe findings from this study have implications for future

investigations related to the employment of PVA-entrapped

GOx with glucose as a new system with antimicrobial activity

of pharmaceutical and technological interest. As regards phar-

maceuticals we have to consider that the increasing develop-

ment of bacterial resistance to traditional antibiotics has

reached alarming levels, thus compelling the strong need to

develop new antimicrobial agents. Last, the observed antibacte-

rial activity of PVA-GOx with glucose could be useful to avoid

the settlement of bacteria which is the primary colonizing pro-

cess in the microbial biofilm development in biomedical

implants.

The proposed polymer/enzyme composite material is struc-

turally simple employing only GOx as biological system.

Nevertheless its bactericide action via a lysozyme-like mecha-

nism has been demonstrated. Further investigations are in

progress, exploiting the noteworthy potential of infrared

spectroscopy, in order to elucidate the linkage between

enzyme-induced generation of reactive oxygen species and lytic

activity. Interestingly our results allowed highlighting the

intrinsic self-disinfecting properties of GOx-based glucose bio-

sensors, when PVA is used as enzyme-entrapping polymer.

This means that PVA-GOx composite materials are particularly

suitable to be employed in analytical applications, such as

implantable sensor devices, where a long-term aseptic contact

with probe and sample is needed. Taking into account that

GOx-synthetic polymer materials have played important roles

in the development of biomaterials for sensing applications in

vivo, our results appear of noteworthy interest in order to

FIGURE 8 Standard assay on Petri dishes with Micrococcus lyso-

deikticus cell walls to detect the lysozyme-like activity of PVA-GOx

in the presence of glucose 10 mM (B), PVA-GOx without glucose

(C), empty wells as control (A), PVA-GOx in the presence of H2O2

1.5 mM (D), only H2O2 1.5 mM (E) and PVA (F). Note the cleared

zone around the wells indicated by the arrows.

Development and Characterization of a Novel Bioactive Polymer 469

Biopolymers

further characterize these systems and assess their real applica-

tive potential in biomedical devices.

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