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Ultrasound assisted lipase catalyzed synthesis of poly-6-hydroxyhexanoate
A. M. Gumel,
1
M. S. M. Annuar,
1*
Y. Chisti,
3
T. Heidelberg
2
1
Institute of Biological Sciences,
2
Department of Chemistry,
Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
3
School of Engineering, PN 456, Massey University, Private Bag 11 222, Palmerston
North, New Zealand
*
Corresponding author:
Dr. M. Suffian M. Annuar
Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia
e-mail: suffian_annuar@um.edu.my
Tel: +60379674003
Fax: +60379674178
Ultrasonics Sonochemistry 19 (2012) 659667
2
Abstract
Ultrasonic irradiation greatly improved the Candida antarctica lipase B mediated ring
opening polymerization of c-caprolactone to poly-6-hydroxyhexanoate in the ionic liquid
1-ethyl-3-methylimidazolium tetraflouroborate. Compared to the conventional
nonsonicated reaction, sonication improved the monomer conversion by 63% and
afforded a polymer product of a narrower molecular weight distribution and a higher
degree of crystallinity. Under sonication, the polydispersity index of the product was
~1.44 compared to a value of ~2.55 for the product of the conventional reaction. With
sonication, nearly 75% of the monomer was converted to product, but the conversion
was only ~16% for the reaction carried out conventionally. Compared to conventional
operation, sonication enhanced the rate of polymer propagation by >2-fold and the
turnover number of the lipase by >3-fold.
Keywords: lipase; polycaprolactone; polyhydroxyalkanoates; ultrasound; kinetics;
biopolymers
Ultrasonics Sonochemistry 19 (2012) 659667
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1. Introduction
This work is concerned with enzyme-mediated sonication-enhanced production of the
biodegradable polymer poly-6-hydroxyhexanoate, a polyhydroxyalkanoates (PHA).
Polymers such as PHA are important in many industrial and biomedical applications
because of their biocompatibility, biodegradability and attractive mechanical properties
[1, 2]. Temporary prostheses, controlled drug delivery devices, resorbable implants and
tissue engineering scaffolds are some of the products made from biodegradable
polymers [3]. Poly-6-hydroxyhexanoate (poly-c-caprolactone) is a semi crystalline
polymer that can be inexpensively produced by ring-opening polymerization (ROP) of
the monomer c-caprolactone [4, 5].
Ring-opening polymerization of lactones and lactides can be achieved with
conventional chemical catalysis or by using biocatalysts [6]. Chemical catalysis relies on
organometallic compounds [7]
that can contaminate the product and are potentially toxic
in biomedical applications. In contrast, the products of enzyme mediated biocatalytic
ring opening polymerization have little or no potential for an adverse health impact. In
addition, enzymatic biocatalysis offers an excellent enantiomeric selectivity, specificity
and catalytic activity under mild reaction conditions. Enzyme catalyzed ROP can make
use of a wide range of substrates including cyclic lactones. The lipase B enzyme of
Candida antarctica has proved to be a highly effective catalyst for ROP of cyclic
lactones [8-11].
Enzyme mediated synthesis of PHAs by ring opening polymerization is
conventionally carried out in potentially hazardous volatile organic solvents such as
Ultrasonics Sonochemistry 19 (2012) 659667
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tetrahydrofuran [12, 13]. An alternative is to use nonvolatile and highly thermostable
ionic liquids as solvents. PHAs and the corresponding monomers tend to be highly
soluble in ionic liquids [4]. Furthermore, enzymes such as lipases have a longer lifespan
in ionic liquids than in conventional organic solvents [4, 14].
Earlier studies of lipase mediated ROP were characterized by poor stability of the
enzyme, a slow rate of reaction, and a low molecular weight of the polymer product.
These problems continue to limit large-scale commercial use of lipase mediated ROP
[15]. Attempts have been made to improve the performance of lipase mediated ROP
through the use of microwave irradiation [16, 17], supercritical fluids [18], and ionic
solvents [12, 19, 20]. Use of ultrasound holds a substantial potential for enhancing the
performance of biocatalytic processes [21], but sonication has not been evaluated for
lipase mediated ROP. This paper reports on ultrasound-assisted lipase-catalyzed ROP
of c-caprolactone to poly-6-hydroxyhexanoate and aims to study its kinetics relative to
the non-sonicated process. The product conversion yield and its selected characteristics
were also studied in comparison with the non-sonicated treatment. The reaction is
carried out in a nonconventional ionic liquid.
2. Materials and Methods
2.1 Materials
All chemicals used were of analytical grade, unless otherwise stated. The ionic solvent
1-ethyl 3-methylimidazolium tetraflouroborate [Emim][BF
4
] (purity >99%) and c-
caprolactone monomer were obtained from Merck (www.merck.com). Standard poly-c-
caprolactone of different molecular weights, tetrahydrofuran, chloroform and methanol
Ultrasonics Sonochemistry 19 (2012) 659667
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were purchased from Sigma-Aldrich (www.sigmaaldrich.com). Candida antarctica lipase
B 435 was purchased from Novozymes (www.novozymes.com). The enzyme was
supplied immobilized on beads of a macroporous acrylic resin [22].
2.2 Methods
2.2.1 Enzymatic ring opening of c-caprolactone
Reactions were performed in triplicate. [Emim][BF
4
] (6.54 M) and c-caprolactone (9.02
M) of known water activity were mixed in 20 mL capped reaction vials. Lipase B (1.5%
w/v, g/100 mL) was added to initiate the reaction except in the primary control vials. The
initial water content and water activity of each reaction mixture were measured using
Metrohms
1
h
1
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
c
-
C
a
p
r
o
l
a
c
t
o
n
e
c
o
n
v
e
r
s
i
o
n
(
%
)
0
10
20
30
40
50
60
70
80
Productivity (not sonicated)
Productivity (sonicated)
Conversion (not sonicated)
Conversion (sonicated)
Figure 5
Ultrasonics Sonochemistry 19 (2012) 659667
37
Time (h)
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
M
n
(
g
m
o
l
1
)
0
2000
4000
6000
8000
10000
12000
M
n
(not sonicated)
M
n
(sonicated)
Time (h)
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
M
w
(
g
m
o
l
1
)
0
5000
10000
15000
20000
25000
M
w
(not sonicated)
M
w
(sonicated)
a)
b)
Figure 6
Ultrasonics Sonochemistry 19 (2012) 659667
38
[E]
0
(g L
1
)
6 8 10 12 14 16 18 20 22
v
p
x
1
0
5
(
m
o
l
L
1
s
1
)
1
2
3
4
5
6
7
8
Sonicated
Not sonicated
[E]
0
(g L
1
)
6 8 10 12 14 16 18 20 22
c
-
C
a
p
r
o
l
a
c
t
o
n
e
c
o
n
v
e
r
s
i
o
n
(
%
)
0
10
20
30
40
50
60
70
80
Sonicated
Not sonicated
a)
b)
Figure 7