POLYMER

TESTING
Polymer Testing 26 (2007) 257261
Material Behaviour
Enzymatic degradation of poly (e-caprolactone) and cellulose
acetate blends by lipase and a-amylase
M.R. Calil

, F. Gaboardi, M.A.G. Bardi, M.L. Rezende, D.S. Rosa

Programa de Pos-Graduac- ao Stricto Sensu em Engenharia e Ciencia dos Materiais (PPG-ECM), Laboratorio de Polmeros Biodegradaveis e
Soluc- oes Ambientais, Universidade Sao Francisco, Rua Alexandre Rodrigues Barbosa, n
%
o. 45, Centro, CEP 13251-900, Itatiba, SP, Brazil
Received 11 July 2006; accepted 23 October 2006
Abstract
Films of poly(e-caprolactone) (PCL) and cellulose acetate (CA) containing different proportions of PCL/CA (100/0, 80/
20, 60/40, 40/60, 20/80 and 0/100, w/w) were prepared by casting, and their degradation by two enzymes (lipase and
a-amylase) was monitored by mass loss and light microscopy for 240 days. Signicant degradation was seen only in
samples digested with lipase. Lipase selectively degraded PCL, with the rate of degradation being inversely proportional to
the CA content. Light microscopy indicated some changes in morphology in the samples after digestion with lipase, and a
relationship between these changes and the degree of mass retentionthe larger the percentage of PCL in blends, the
greater the loss of mass. However, there was no difference in the morphology among the samples incubated with
a-amylase. The results suggested that lipase acts selectively on PCL, with little effect on CA.
r 2006 Published by Elsevier Ltd.
Keywords: a-amylase; Blend degradation; Cellulose acetate; Enzymatic degradation; Lipase; Poly(e-caprolactone); Polymer blends
1. Introduction
There is increasing concern in society about the
accumulation of plastic-waste materials and their
potentially harmful environmental effects [1]. An
alternative to reduce the impact of these materials is
to use biodegradable polymers [2], the properties of
which are similar to or better than those of
conventional plastics [3]. Under appropriate envir-
onmental conditions, biodegradable polymers can
be degraded into simpler compounds that can be
incorporated into the carbon and nitrogen cycles [4].
This degradation is mediated largely by enzymes
produced by microorganisms [5].
Among biodegradable polymers, poly(e-caprolac-
tone) (PCL), a synthetic aliphatic polyester, has
been widely used in medical, packaging and
agricultural applications because of its excellent
mechanical properties, including its exibility [5].
The major disadvantage of PCL is its price, which
limits its wider use as a substitute for conventional
polymers.
Polymeric blends, i.e., mixtures of two or more
polymers that may or may not be biodegradable, are
commonly used in the plastic industry [6]. In
particular, blends of PCL and natural materials,
such as starch and cellulose derivatives [7] have been
extensively studied because of their lower cost
compared to other materials [8].
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0142-9418/$ - see front matter r 2006 Published by Elsevier Ltd.
doi:10.1016/j.polymertesting.2006.10.007

Corresponding author. Tel.: +55 11 4534 8046;

fax: +55 11 4524 1933.
E-mail address: mrcalil@terra.com.br (M.R. Calil).
Cellulose, a natural polymer produced by plants,
is a polysaccharide composed of long chains of
glucose molecules. This polymer can be chemically
modied to produce cellulose acetate (CA), the
properties of which are directly inuenced by the
degree of substitution by acetate groups [9]. CA is a
semicrystalline polymer having excellent mechanical
properties. Blends of CA with PCL have a reduced
cost and show biodegradation [10].
Many methods are currently used to assess the
biodegradability of polymeric systems, including
ageing tests in the presence of enzymes, i.e.,
accelerated (enzyme-catalyzed) degradation [1114].
Enzymatic degradation may involve simple or multi-
ple enzyme systems, with the enzymes being synthe-
sized by microorganisms. Enzymes reduce the energy
of activation and weaken the chemical bonds in the
polymer, thereby decreasing the energy required for
degradation. Factors such as temperature and pH
can affect the enzymatic activity [15], although the
rate of degradation also depends on the character-
istics of the system components [16].
Lipases are hydrolases that cleave ester bonds to
release fatty acids and glycerol. These enzymes are
common in plant and animal tissues and can also be
produced by microorganisms during fermentation
[17]. In contrast, a-amylase is an enzyme that
hydrolyses glycosidic bonds.
Gan et al. [18] reported that PCL was easily
degraded by lipases from microorganisms, espe-
cially Pseudomonas spp. Similarly, Marten et al. [19]
also studied the effect of enzymes on polyesters.
Li et al. [16] described a system to study the
biodegradation of PCL and poly (L-lactide) blends
using a Pseudomonas sp. lipase. The addition of
poly (L-lactide) to PCL markedly reduced the
degradation of the former polymer. In this system,
the presence of cracks and an elevated lipase
concentration favoured enzymatic degradation.
Sivalingam et al. [20] studied the enzymatic biode-
gradation of PCL by two enzymes, novozyme 435
and lipolase, and found that there was less
degradation with the former enzyme than with
lipolase.
Braganc-a [21] reported that hydrolysis was the
principal mechanism for the biodegradation of CA,
with the rst step as depolymerization and being in
contact with extracellular microbial enzymes; the
resulting oligomers were then easily phagocytosed
by the cells, followed by mineralization.
Lipase is often used in the biodegradation of
aliphatic polyesters. Tserki et al. [22] studied the
biodegradation of several polyesters by lipases from
Candida cylindracea and Rhizopus delemar, and
cholesterol esterase from Pseudomonas uorescens.
Their results showed that the degree of crystallinity
directly affected the degradation of the polymer.
The aim of this work was to examine the
enzymatic degradation of PCL and CA blends by
lipase and a-amylase. The extent of degradation was
assessed by measuring the mass retention and by
monitoring the morphological changes using light
microscopy.
2. Experimental
2.1. Materiais
PCL (type P-767) was supplied in pellet form by
Dow Qu mica S.A. (Cubata o, SP, Brazil). The
melt ow at 80 1C was 1.970.3 g/10 min (ASTM
D-1238), with a density of 1,145 kg/m
3
and a
weight average molecular weight (Mw) of 50,000.
CA (type 15-4051) was supplied in ake form by
Rhodia Acetow Brazil Ltda. (Santo Andre , SP,
Brazil). The average degree of substitution was
2.4, the degree of polymerization was 500600
and the specic viscosity was 40.295.
2.2. Film preparation
The PCL/CA blends were prepared by casting
using the following proportions of PCL/CA (w/w):
80/20, 60/40, 40/60, and 20/80. Pure materials were
represented by 100/0 and 0/100 for PCL and CA,
respectively. The solutions were prepared by dissol-
ving the material in 10% (w/v) acetone, with stirring
at 6075 1C for 6 h. The mixtures were then poured
into culture dishes and the solvent was allowed to
evaporate in an atmosphere saturated with acetone.
2.3. Ageing in medium with lipase
Each sample was placed in a vial lled with 10 ml
of 0.05 M phosphate buffer, pH 7.4, containing
1.0 mg of lipase and 0.02% sodium azide, and then
incubated in a thermostatted oven at 37 1C. The
buffer/enzyme system was changed for every 24 h
during the evaluation period in order to maintain
the original level of enzymatic activity. For
every 48 h, the samples were removed from the
incubation medium, washed with distilled water,
wiped dry, weighed, and examined by light micro-
scopy before being returned to the incubation
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M.R. Calil et al. / Polymer Testing 26 (2007) 257261 258
medium. The controls consisted of samples incu-
bated in buffer without enzyme.
2.4. Ageing in medium with a-amylase
Each sample was placed in a vial lled with 10 ml
of 0.05 M acetate buffer, pH 6.0, containing 2.7 mg
of enzyme, according to the Corn Products proto-
col. The vials were placed in a water bath at 60 1C
and for every 48 h the samples were removed,
washed with distilled water, wiped dry, weighed,
and examined by light microscopy before being
returned to the incubation medium. The controls
consisted of 10 ml of acetate buffer without enzyme.
2.5. Light microscopy
The morphology and degree of phase separation
of the PCL/starch blends were assessed by light
microscopy (model XP-500 LABORANA, Sa o
Paulo, SP, Brazil), with a magnication of 20 .
3. Results and discussion
3.1. Evaluation of degradation based on mass
retention
Figs. 1 and 2 show the mass retention of the pure
polymers (PCL and CA) and their blends during
ageing with lipase and a-amylase, respectively.
Signicant degradation was seen only in samples
digested with lipase. This nding reected the
specicity of lipase for PCL, as described by Rosa
et al. [14]. Pure PCL showed the highest loss of mass
when compared to pure CA and to PCL/CA blends.
The same behaviour was also observed by Calil
et al. [13] when PCL, CA and PCL/CA blends were
submitted in a mixture of fungi on solid and liquid
medium. Pure CA showed an increase in mass in
both the enzymatic systems, and this likely reected
the strong interactions among the CA chains that
did not allow water molecules to access to the
free hydroxyl groups in CA. Another fact to be
considered is the hydrophobic nature of CA, which
could have prevented a more efcient hydrolysis.
Although CA is chemically simple, its structure is
complex, and often requires the action of various
enzymatic complexes to ensure complete degrada-
tion of the material.
In the blends, the extent of degradation was
inversely proportional to the CA content. Braganc-a
[21] reported that the addition of CA increased
the crystallinity of PCL and made the enzymatic
biodegradation of these polymers more difcult.
Studies of cellulose derivatives have shown that the
crystallinity and structural organisation of cellulose
vary according to its processing and that structural
modications of cellulose inuence its biodegrada-
tion. Finally, the linear and apolar structure of
cellulose means that this polymer is not very
amenable to hydrolysis during biodegradation.
3.2. Light microscopy
Fig. 3 shows the initial and nal morphology of
samples incubated with lipase and a-amylase.
ARTICLE IN PRESS
0 50 100 150 200 250
50
60
70
80
90
100
110
PCL/AC
100/0
80/20
60/40
40/60
20/80
0/100
M
a
s
s

r
e
t
e
n
t
i
o
n

(
%
)
Time (h)
Fig. 1. Mass retention for the PCL/CA formulations submitted
to the action of the enzyme lipase.
0 50 100 150 200 250
96
98
100
102
104
106
108
110
112
114
116
118
M
a
s
s

r
e
t
e
n
t
i
o
n

(
%
)
Time (h)
PCL/AC
100/0
80/20
60/40
40/60
20/80
0/100
Fig. 2. Mass retention for the PCL/CA formulations submitted
to the action of the enzyme a-amylase.
M.R. Calil et al. / Polymer Testing 26 (2007) 257261 259
Incubation with lipase resulted in the appearance
of small ssures in the 100/0 and 80/20 blends, and
this change was probably related to the mass
retention of the samples, since these two formula-
tions showed the highest loss of mass (Fig. 1). In
contrast, samples incubated with a-amylase showed
no signicant changes in morphology, and this was
reected in the low loss of mass, as also observed by
Lopes [23]. In general, there was a relationship
between the rate of biodegradation and the mis-
cibility of the material, with samples containing
440% PCL showing the greatest morphological
changes. According to Lopes [23], low miscibility
and consequently, low-molecular adhesion facilitate
the enzymatic attack of polymers and enhance their
(bio)degradation.
4. Conclusions
The results of this study indicate that lipase acts
selectively on PCL, with little effect on CA. The low
biodegradation of CA probably reects the hydro-
phobic nature of this polymer and the fact that it is
not a specic substrate of the enzymes used. The
greater the amount of PCL added to the blend, the
greater the degradation of the samples by lipase, a
nding corroborated by the extent of morphological
damage seen by light microscopy. In contrast to
these ndings, a-amylase had no signicant effect on
the structure and morphology of the samples. In
addition, the results suggest that of the two enzymes
studied only lipase can degrade PCL and that it
cannot degrade CA.
Acknowledgements
This work was supported by FAPESP (grant nos.
02/13202-06 and 04/13359-8), CNPq (grant nos.
477942/2003-2, 362240/2004-3 and 304577/2004-9)
and Universidade Sa o Francisco. The authors also
thank Union Carbide Ltd. for supplying of the
PCL, and Corn Products Brazil for supplying the
a-amylase.
References
[1] S.Y. Lee, Plastic bacteria? Progress and prospects for
polyhydroxyalkanoate production in bacteria, Trends Bio-
technol 14 (11) (1996) 431438.
[2] D.S. Rosa, B.L.M. Franco, M.R. Calil, Biodegradabilidade
e propriedades, Pol meros: Cie ncia Tecnol 11 (2) (2001)
8288.
[3] M.E. Gomes, A.S. Ribeiro, P.B. Malafaya, R.L. Reis, A.M.
Cunha, A new approach based on injection molding to
produce biodegradable starch-based polymeric scaffolds:
morphology, mechanical and degradation behavior, Bioma-
terials 22 (9) (2001) 883889.
[4] Y.S. Chun, Y.J. Kyung, H.C. Jung, W.N. Kim, Thermal and
rheological properties of poly(e-caprolactone) and polystyr-
ene blends, Polymer 41 (24) (2000) 87298733.
[5] D.S. Rosa, P.R. Filho, Biodegradac-a o: um ensaio com
pol meros, Moara, Itatiba, 2003.
[6] E.B. Mano, L.C. Mendes, Introduc- a o a pol meros, Edgard
Blu cher, Sa o Paulo, 1999.
[7] K. Kuwabara, Z. Gan, T. Nakamura, H. Abe, Y. Doi,
Molecular mobility and crystalline phase structure of
biodegradable poly(3-hydroxybutylic acid-co-3-hydroxyhex-
anoic acid), Polym. Degradat. Stabil. 84 (1) (2004) 135141.
[8] L. Rouxhet, R. Legras, Modications induced by swift
heavy ions in poly(hydroxybutyrate-hydoxyvalerate) (PHB/
HV) and poly(e-caprolactone) (PCL) lmsThermal beha-
vior and molecular mass modication, Nucl. Instrum.
Methods Phys. Res. B: Beam Interactions Mater. Atoms
171 (4) (2000) 487498.
[9] M. Ita vaara, M. Siika-aho, L. Viikari, Enzymatic degrada-
tion of cellulose-based materials, J. Environ. Polym. Degrad.
7 (2) (1999) 6773.
ARTICLE IN PRESS
PCL/CA
Formulations
Initials
Samples
submitted to
lipase
Samples
submitted to -
amylase
100/0
80/20
60/40
40/60
20/80
0/100
Fig. 3. Photomicrographs of the PCL/CA formulations sub-
mitted to the action of the enzymes lipase and a-amylase, after
240 days.
M.R. Calil et al. / Polymer Testing 26 (2007) 257261 260
[10] T. Hirotsu, A.A.J. Ketelaars, K. Nakayama, Biodegradation
of poly(e-caprolactone)-polycarbonate blend sheets, Polym.
Degradat. Stabil. 68 (3) (2000) 311469.
[11] D. Preechawong, M. Peesan, P. Supaphol, R. Rujiravanit,
Preparation and characterization of starch/poly(L-lactide
acid) hybrid foams, Carbohydrate Polym. 59 (3) (2005)
329337.
[12] M. Vikman, M. Ita vaara, K. Poutanem, Biodegradation of
starch-based materials, Marcel Dekker, New York, 2006.
[13] M.R. Calil, F. Gaboardi, C.G.F. Guedes, D.S. Rosa,
Comparison of the biodegradation of poly(e-caprolactone),
cellulose acetate and their blends by the Sturm test
and selected cultured fungi, Polym. Testing 25 (2006)
597604.
[14] D.S. Rosa, D.R. Lopes, M.R. Calil, Thermal properties and
enzymatic degradation of blends of poly(e-caprolactone)
with starches, Polym. Testing 24 (6) (2005) 756761.
[15] R. Chandra, R. Rustgi, Biodegradable polymers, Prog.
Polym. Sci. 23 (7) (1998) 12731335.
[16] S. Li, L. Liu, H. Garreau, M. Vert, Lipase-catalyzed
biodegradation of poly(e-caprolactone) blends with various
polylactide-based polymers, Biomacromolecules 4 (2) (2003)
372377.
[17] D.K. Gilding, Biodegradable polymers, Biocompatibil. Clin.
Implant Mater. 11 (2) (1981) 209232.
[18] Z. Gan, D. Yu, Z. Zhong, Q. Liang, X. Jing, Enzymatic
degradation of poly(e-caprolactone)/poly(DL-lactide) blends
in phosphate buffer solution, Polymer 40 (10) (1999)
28592862.
[19] E. Marten, R.-J. Mu ler, W.-D. Deckwer, Studies on
hydrolysis of polyesters. IIAliphatic-aromatic copolye-
sters, Polym. Degradat. Stabil. 88 (3) (2005) 371381.
[20] G. Sivalingam, S. Chattopadhyay, G. Madras, Solvent
effects on the lipase catalyzed biodegradation of poly(e-
caprolactone) in solution, Polymer Degradation and Stabi-
lity 79 (3) (2003) 413418.
[21] F.C. Braganc- a, Desenvolvimento de blendas de poli(e-
caprolactona) e acetato de celulose e suas propriedades
meca nicas, te rmicas, morfolo gicas e de biodegradac- a o,
Universidade Sa o Francisco, Itatiba, 2003.
[22] V. Tserki, P. Matzinos, E. Pavlidou, D. Vachliotis,
C. Panayiotou, Biodegradable aliphatic polyesters. Part I
Properties and biodegradation of poly(butylenes succinate-
co-butylene adipate), Polym. Degradat. Stabil. 91 (2) (2006)
367376.
[23] D.R. Lopes, O efeito da estrutura qu mica de diferentes
amidos e plasticantes nas propriedades meca nica, te rmica,
morfolo gica e de biodegradac- a o das blendas de amido e
poli(e-caprolactona), Tese de mestrado, Universidade Sa o
Francisco, Itatiba, 2003.
ARTICLE IN PRESS
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