Lipase-Catalyzed Synthesis and Characterization of

Biodegradable Polyester Containing L-Malic Acid Unit in

Solvent System

Dahu Yao,1,2 Guangji

1,7
Li,3 Tapas Kuila,2 Peng Li,4 Nam Hoon Kim,5 Seong-Il Kim,6
Joong Hee Lee
1
BIN Fusion Research Team, Fusion Research Team, Department of Polymer and Nano Engineering, Chonbuk
National
2
University, Jeonju, Jeonbuk, South Korea
College of Chemical Engineering and Pharmaceutics, Henan University of Science and Technology, Luoyang,
People’s Republic of China
3
College of Material Science and Engineering, South China University of Technology, Guangzhou,
People’s Republic of China
4
5
College of Chemistry and Chemical Engineering, China University of Petroleum, Qingdao, People’s Republic of China
6
Department of Hydrogen and Fuel Cell Engineering, Chonbuk National University, Jeonju, Jeonbuk, South Korea
Department of Technology Education, Daebul University, Jeonnam, Republic of Korea
7
Department of BIN Fusion Technology, Chonbuk National University, Jeonju, Jeonbuk, South Korea

Received 24 March 2010; accepted 21 August 2010

DOI 10.1002/app.33257
Published online 8 November 2010 in Wiley Online Library (wileyonlinelibrary.com).

ABSTRACT: Lipase-catalyzed direct polycondensation of L- L-MA feed ratio in the diacids of 40 mol %. The Mw
malic acid (L-MA), adipic acid, and 1,8-octanediol in or- increased from 3.2 to 16.6 kDa when the reaction time was
ganic media was achieved using Novozym 435 as the bio- extended from 6 to 48 hr at 70 ○C, and remained relatively
catalyst. 1H-nuclear magnetic resonance spectroscopy
constant with further increases in reaction time from 48 to
indicated that the selectivity of Novozym 435 was unaf-
fected by changes in the organic media. The molecular 72 hr. The hydrophilicity, thermal stability, and crystalliz-
ability of the copolymer were also investigated. VC
weight (Mw) of the copolymers was affected by the L-MA 2010 Wiley Periodicals, Inc. J Appl Polym Sci 120: 1114–1120, 2011
feed ratio in the diacids, hydrophobicity of the solvent,
and solubility of the substrates in the solvents. The Mw Key words: polyesters; biodegradable; polycondensation;
reached a maximum of 17.4 kDa at 80○C in isooctane at a enzymes

INTRODUCTION that could be used to tailor the physical properties and

As one of the most important classes of synthetic bio- introduce bioactive substances. Compared with con-
materials, many aliphatic polyesters have good biocom- ventional aliphatic polyesters, poly(malic acid) and pol-
patibility and biodegradability and have attracted yesters containing L-malic acid (L-MA) units contain
increasing attention in biomedical fields, such as tissue many pendant carboxyl- or hydroxyl-functional groups
engineering, surgical sutures, gene therapy, and con- along the macromolecular chains and have attracted
1 considerable attention.2–6 The chemical properties of
trolled drug delivery. However, the applications of
conventional aliphatic polyesters are limited because of copolymers, such as their hydrophobic/hydrophilic
their hydrophobicity and absence of functional groups balance,7,8 degradation rate,9–11 modifiability, etc.,12,13 can
be adjusted for different applications14–16 by chang- ing
the L-MA content or varying the chemical structures of
the pendant groups.
There are many reports on chemical routes to synthe-
Correspondence to: J. H. Lee ([email protected]) or G. Li
([email protected]). size functional polyesters containing L-MA units by
Contract grant sponsor: National Space Laboratory ring-opening polymerization (ROP) and polycondensa-
(NSL) Program; contract grant number: S1 08A01003210. tion.17,18 Amphiphilic poly[(R,S)-b-malic acid-b-e-capro-
Contract grant sponsor: Human Resource Training lactone] diblock copolymers were synthesized by the
Project for Regional Innovation.
ROP of (R,S)-b-benzyl malolactonate with e-caprolac-
Contract grant sponsor: World Class University (WCU)
Program, Ministry of Education, Science and Technology tone, followed by removing the benzyl ester protecting
(MEST); contract grant number: R31-20029. group by catalytic hydrogenolysis.7,8 Wang and co-
workers9,10 synthesized a functional polylactide copoly-
Journal of Applied Polymer Science, Vol. 120, 1114–1120 mer with carboxyl acid side chains using a similar
(2011) method. Zhang et al.18 synthesized poly(butylenes
VC 2010 Wiley Periodicals, Inc.
BIODEGRADABLE POLYESTER CONTAINING L-MA 111
5

succinate-co-butylene malate) with free hydroxyl pend- TABLE I

ant groups by the polycondensation of dimethyl Effect of Solvents and Temperature on the Mw and Mw/
malate, 1,4-butanediol, and succinic acid. The entire Mn without Lipase
process involves four steps, including protection and Entry Organic solvent T (○C) Mw (×103) PDI
deprotection steps of hydroxyl of dimethyl malate and
repeated purification of intermediates. 1 Isooctane 60 1.3 1.17
2 n-Hexane 60 1.5 1.19
Common difficulties encountered during the syn-
3 Toluene 60 1.6 1.21
thesis of functional polyesters containing L-MA using 4 Toluene 70 1.9 1.29
conventional methods include the following: 5 Toluene 80 2.0 1.24
(1) protection–deprotection steps lead to a lower 6 Chloroform 60 0.8 1.07
yield, (2) repeated purification of intermediate con- 7 t-BuOH 60 0.9 1.10
sumes a large amount of organic solvents, resulting 8 THF 60 1.1 1.15
in environmental problems, (3) difficulty in achiev- 9 Acetone 60 0.8 1.04
ing complete deprotection of the functional groups,
The feed molar ratio was OC : ADA : L-MA 15 :¼9 : 6. The
and (4) toxicity of the residual organometallic com- reaction time was 48 hr. All the procedures are same with
pounds used as polymerization catalysts. the procedure for the enzyme-catalyzed copolymer- ization
In vitro enzyme-catalyzed synthesis of polyesters described in the article. PDI, polydispersity index of the
is a new and eco-friendly process.19–22 Functional polymer.
polyesters with different functional pendant groups round-bottom flask. The flask was capped with a
can be synthesized simply by enzyme-catalyzed syn- rubber septum and placed into an oil bath main-
thesis because of its high selectivity. 23–25 The diffi- tained at 120○C for 3 hr with magnetic stirring. The
culties encountered in synthesizing functional poly- temperature of the oil bath was then decreased to
esters containing L-MA by conventional methods can the prescribed temperature (90–50○ C), and 4-A˚ mo-
be resolved by enzyme-catalyzed synthesis. Previous lecular sieves (to absorb the water producing in the
work26,27 investigated the lipase-catalyzed direct reaction), different organic solvents, and Novozym
polycondensation of L-MA, adipic acid (ADA), and 435 (10% by weight based on total monomers, dried
1,8-octanediol (OC) for preparing polyesters contain- in vacuum desiccator under 10 mmHg at 25○C for 24
ing L-MA units in a solvent-free system. However, hr) were added to the flasks. The flask was sealed,
for enzyme catalysis in nonaqueous medium, and the magnetic stirring rate was set to 200 rpm.
organic solvents play an important role.19 The selec- The reaction was quenched by adding an excess
tivity, catalytic activity, and stability of enzyme can of
be altered by proper selection of the organic solvent. chloroform with constant stirring for 15 min after 48
The solvent also can regulate the partitioning of sub- hr. The Novozym 435 and 4-A˚ molecular
strates and products between the solvent and the sieves were removed by filtration. The filtrate was
enzyme.19 Therefore, this study examined the effect of added
different reaction conditions on the lipase-cata- lyzed slowly into a flask containing an excess of cold n-
direct polycondensation of comonomers L-MA, ADA, hexane with rapid stirring, and the resulting product
and OC in a solvent system. was isolated by centrifuging. The product was dried
in vacuum at 50○C for 48 hr. The control synthesis
EXPERIMENTAL was done without lipase in different organic solvents
(the feed molar ratio is OC : ADA : L-MA ¼ 15 : 9 :
Materials 6), and the results are shown in Table I.
OC (98% purity) was purchased from Aldrich
Chemical Co. (St. Louis, MO). L-MA (98% purity) was
Characterization
obtained from Guangzhou Huangkai Co. (Guangzhou,
China). Isooctane, n-hexane, toluene, chloroform, t- Structural characterization
butanol, tet- rahydrofuran (THF), acetone, and ADA The structures of the prepared products were char-
(Guangzhou Chemical Co.) were of analytical grade. acterized by nuclear magnetic resonance (NMR)
All chemicals were used as received. Novozym 435 spectroscopy. The 1H-NMR spectra were recorded
(immobilized lipase from Candida antarctica, type B, on a Bruker AV 300 spectrometer operating at 300.1
10,000U g—1) was purchased from Novozymes MHz. The polyesters were dissolved in deuterated
(Denmark). chloroform. Tetramethylsilane was used as the inter-
nal standard (d ¼ 0.00 ppm).
Enzyme-catalyzed polymerizations of L-MA
with OC
Molecular weight measurements
OC (2.193 g, 15 mmol) was mixed with a mixture of
The molecular weight and polydispersity were
total of 15 mmol of L-MA and ADA in a 100-mL
determined by gel permeation chromatography using
the basis of conventional calibration curve
Journal of Applied Polymer Science DOI 10.1002/app
 ucts between the solvent and the enzyme. Thus, the
selectivity, catalytic activity, and stability of enzyme
are altered by the organic solvents.

Structural characterization
Figure 1 shows the 1H-NMR spectra of poly(octylene
adipate), and Figure 2 shows the 1H-NMR spectra of
copolyesters containing the L-MA units synthesized in
different organic media. The resolved signals for the
OC units protons CH2AO(C¼¼O), 1, and CH2AOH,
10 , were observed at 4.07 and 3.65 ppm from 1 the H-

NMR spectrum of poly(octylene adipate),

respectively. The signals attributed to the methylene
protons CH2A(C¼¼O), 5, and CH2A(C¼¼OOH), 50 ,
of the ADA units were not resolved and appear as a
multiplet from
Figure 1 1H-NMR spectrum of poly(octylene adipate). 2.25 to 2.45 ppm. The signals for the protons CH CH
AO(C¼¼O),
2 2 2, and CH CH AOH,2 220 , of the OC
generated by narrow molecular weight polystyrene units, and ADA units protons CH2CH2A(C¼¼O), 6,
standards. Gel permeation chromatography analyses and CH2CH2A(C¼¼O)OH, 60 , all appear as a broad
were performed at 35○C using a Shimadzu LC-10 multip- let from 1.57 to 1.78 ppm. Similarly, the
HPLC System equipped with a LC-10AD pump, a signals corre- sponding to the methylene protons,
SIL-10A auto sampler, a differential refractometer CH2CH2CH2AO (C¼¼O), 3, and
(RI detector), and a series of Agilent columns (three CH2CH2CH2CH2AO(C¼¼O), 4, of the OC units were
PLgel 5 lm MIXED-D and -E columns). THF was unresolved and observed between 1.24 and 1.45
used as the eluent at a flow rate of 1.0 mL/min. ppm.23,26,27 The 1H-NMR spectra of the copo- lyesters
containing the L-MA units synthesized in dif- ferent
solvents (Fig. 2) showed signals corresponding to the
Thermal analysis
L-MA units protons CHOH(C¼¼O), 7, and
Differential scanning calorimetry (DSC) analyses CH2(C¼¼O), 8, at 4.5 ~ 4.6 and 2.7 ~ 2.9
were carried out using a DSC 204 F1 differential ppm.18,26,27
scanning calorimeter from Netzsch Instruments. The There was no reaction between the hydroxyl groups of
samples (8–14 mg) were heated from —70 to þ100○C L-MA and the carboxyl groups of ADA or L-MA
at a rate of 10○ C/min with a nitrogen purge. Ther- according to 1H-NMR. The MA units were incorpo-
mogravimetric analysis was carried out using a TG rated into the macromolecular chains exclusively
209 thermogravimetric analyzer from Netzsch through their carboxyl groups. The copolyesters
Instruments. The samples (12–16 mg) were heated obtained in different solvents were poly(octylene
from ambient temperature to 800○C at a rate of
20○C/min with a nitrogen purge.

Contact angle measurement

The contact angles were measured to determine the
hydrophilicity of the copolymers at 20○C using the
sessile drop method on a contact-angle measurement
apparatus (JY-82; Harke Co., St. Charles, MO). The
static contact angle was measured at a contact time
of t ¼ 30 sec. Drops of liquid were prepared using a
microsyringe and dropped onto the surface of the
polymer films.

RESULTS AND DISCUSSION

Organic solvents play an important role on enzyme
catalysis in nonaqueous medium. The organic sol-
vents change the molecular conformation of enzyme
and regulate the partitioning of substrates and prod- Figure 2 1H-NMR spectra of poly(octylene adipate-co-oc-
tylene malate) synthesized in different solvents: (a) ace-
tone, (b) chloroform, (c) THF, (d) t-BuOH, (e) toluene, (f) n-
hexane, and (g) isooctane.
adipate-co-octylene malate). The copolymers
produced by Novozym 435-catalyzed direct
polycondensation showed no branching through the
esterification of L-MA hydroxyl groups. Instead, the
copolyesters con- taining L-MA units contained
pendant hydroxyl groups that were available for
postproduct modifica- tion. The selectivity of
Novozym 435 was not affected by the organic media.

Effects of solvents
The reaction medium is one of the most important
factors in nonaqueous enzyme catalysis. The enzyme-
catalyzed reaction can be manipulated in a nonaqu-
eous phase by prudently choosing the appropriate
solvent.19,28–30 According to reports on the lipase-cata-
lyzed polyesters by ROP and polycondensation,28–30
the molecular weights of the polymer and conversion
of the monomer in hydrophilic organic solvents were
lower than those in hydrophobic solvents. Enzymatic
polymerization was carried out in seven solvents with
different hydrophobicity (expressed as log P, in which
P was the partition coefficient between 1-octanol and
water) to determine the effects of the organic solvents.
The prepolymer and the copolymer all dissolve in t-
BuOH, THF, acetone, chloroform, and toluene, and
are insoluble in hexane and isooctane. The effect of
different solvents on the molecular weight and poly-
dispersity index without using lipase is summarized
in Table I. As shown in Figure 3, using hydrophilic
solvents (log P < 2, including t-BuOH, THF, and ace-
tone) resulted in lower molecular weights compared
with hydrophobic solvents (log P > 2, including tolu- Figure 3 Effects of solvents on Mw for low content (a) and
high content (b) of L-MA. The diacids feed molar ratios
ene, hexane, and isooctane), which was in accordance (ADA : L-MA) were 6 : 4 for (a) and 0 : 10 for (b). For all
with the results of the lipase-catalyzed ROP of e-cap- experiments, the reaction time was 48 hr, and the reaction
rolactone. This is attributed to the deactivation of temperature was 60○C.
enzyme in hydrophilic media because of enzyme con-
formational changes. Compared with hydrophobic cores of hydroxyl and carboxyl groups surrounded
solvents, hydrophilic solvents tend to strip the essen- by hydrophobic molecular chains. The reactive ter-
tial hydration water from the enzyme, and result in minal groups of the substrates were embedded in
the distortion of catalytic conformation and losing its hydrophilic cores and show poor reactivity. This
catalytic activity partially or even completely. results in a lowering of the molecular weights of the
However, the effects of highly hydrophobic sol- copolymer at a higher L-MA content in toluene. On
vents on the enzymatic polymerization are more the other hand, the molecular weight of copolymer
complex compared with the lipase-catalyzed ROP of is higher in isooctane and n-hexane because the sub-
e-caprolactone. Toluene, hexane, and isooctane are strates are insoluble in these two solvents.
all benign media if the L-MA content in the diacids
in the polymerization system is low [Fig. 3(a)]. If the L-
MA content is high [Fig. 3(b)], hexane and isooc- tane Effects of the L-MA content
are still benign solvents, but toluene is not. This could Table II lists the molecular weight (Mw) and its dis-
be due to the variation of hydrophilicity of the tribution (Mw/Mn) of copolymers prepared by Novo-
substrates (including prepolymer and copolymer) zym 435-catalyzed polycondensation with various L-
with the variation of monomers ratio. The substrates MA contents in the diacids. The Mw of the copoly-
are amphiphilic and soluble in toluene, and their mer decreased with increasing L-MA content in the
hydrophilicity increases with increasing L-MA con- diacids from 0 to 60 mol % and remained relatively
tent. The molecular chains of substrates can easily constant with further increases in concentration. For
form aggregates in toluene-containing hydrophilic example, Mw decreased from 24,200 to 11,500 when
the L-MA content in the diacids was increased from
 TABLE II
The Content of L-MA versus the Enzymatic Polycondensation
Feed molar ratio Molar ratio in polymera
3
Entry Sample OC : ADA : L-MA ADA : L-MA Yield (%) Mw (×10 ) Mw/Mn
1 B1 100 : 100 : 0 100 : 0 97 24.2 2.30
2 B2 100 : 90 : 10 91 : 9 94 21.5 2.10
3 B3 100 : 80 : 20 79 : 21 95 19.8 2.01
4 B4 100 : 70 : 30 73 : 27 92 17.2 2.50
5 B5 100 : 60 : 40 58 : 42 91 16.6 2.42
6 B6 100 : 40 : 60 42 : 58 89 11.5 2.29
7 B7 100 : 20 : 80 23 : 77 87 11.2 2.25
8 B8 100 : 0 : 100 0 : 100 90 10.6 2.18

The organic medium was isooctane, and the isooctane (volume, mL) to total monomer ratio (weight, g) was 2 : 1. The
reaction temperature was 70○C.
a
Determined by 1H-NMR.

0 to 60 mol %, and changed from 11,500 to 10,600 Effects of the reaction temperature and time
with further increases in the L-MA content from 60
to 100 mol %. This suggests that the carboxylic Figures 4 and 5 show the effects of reaction tempera-
groups of L-MA have lower activity than that of ture and time on the lipase-catalyzed polymeriza-
ADA. This is due to the higher hydrophilicity of L- tion, respectively. As shown in Figure 4, the rule of
MA, which makes it difficult to diffuse into the Mw changing with reaction temperature in isooctane
hydrophobic active sites of the enzyme and form a is similar to that in a solvent-free system.27 How-
transition complex.31,32 In addition, copolymers with ever, the Mw in isooctane was higher than that in the
free hydroxyl pendants can form hydrogen bonds, solvent-free system. As shown in Figure 4, the Mw of
and the energy of hydrogen bond of the copolymer the copolymers increased from 8600 to 17,400, with
increases with increasing L-MA content in the increasing temperature from 50○C to 80○C. However,
copolymers. This results in a higher viscous reaction further increases in reaction temperature from 80○C
system and decreases the diffusion rate of the acyl to 90○C led to decrease in Mw from 17,400 to 12,800.
donor to the hydrophobic active sites of the enzyme. The viscosity of the reaction system decreased with
This could restrict the molecular weight of the increasing temperature, which resulted in decreasing
copolymers.33,34 diffusion constraints existing in the lipase-catalyzed
Compared with the solvent-free system, Mw in iso- polymerization reaction.33 Consequently, the Mw of
octane is higher.26,27 This can be explained by the the copolymer is increased with increasing tempera-
strong negative effect of L-MA on the stability of ture. On the other hand, lipase can be denatured,
Novozym 435.32,35 Novozym 435 had higher stability and it will partially or even completely lose its
and activity in isooctane system than those in sol- catalytic activity at higher temperature, which can
vent-free system. result in the corresponding decrease in Mw. The
change in the molecular weight distribution Mw/Mn

Figure 4 Mw and Mw/Mn versus reaction temperature in

isooctane solvent. The diacids feed molar ratios were ADA Figure 5 Mw and Mw/Mn versus reaction time in isooc-
: L-MA ¼ 6 : 4, and the reaction time was 48 hr. tane. The diacids feed molar ratios were ADA : L-MA ¼
6 : 4, and the reaction temperature was 70○C.
 TABLE III
Thermal Properties Obtained from DSC Analyses
Sample Tm (○C) DHf (J/g)
B1 69.3 111.8
B2 63.8 107.5
B3 61.8 77.8
B4 53.5 62.7
B5 48.3 53.6
B6 32.4 32.5
B7 – –
B8 – –

steric hindrance than for long-chain substrates because

of the specific structure of the enzyme binding site.31
As a type of protein, lipase can be denatured with pro-
Figure 6 The contact angle of the copolymers with differ- longation of reaction time, which would also limit the
ent L-MA content. increase in molecular weight of the copolymers.
(polydispersity index) of the copolymers with tem- Contact angle
perature is similar to the change in Mw.
As shown in Figure 5, the Mw of the copolymers Figure 6 shows the relationship between the static
increased from 3200 to 11,900 and 16,600 with increas- water contact angle and the L-MA content in the
ing reaction time from 6 to 24 hr and 48 hr. The Mw monomer feed ratios. The contact angle decreased
changed little when the reaction time was increased with increasing L-MA content. For example, a
from 48 to 72 hr. This may be due to a side reaction, decrease in contact angle from 70.5 ○ to 32○ was
such as hydrolysis, enzyme specificity with the chain observed when the L-MA content in the diacids was
length, and deactivation.19 The concentration of ester increased from 0 to 40 mol %, suggesting that the
bonds increased with increasing molecular weight of hydrophilicity of the copolymer increases with
the copolymers. At the same time, the concentration of increasing L-MA content. This is due to the increase
reactive hydroxyl and carboxyl terminal groups in the density of the pendant hydroxyl groups in
decreased. The hydrolysis of ester bonds increased and copolymers with increasing L-MA content. The
esterification decreased accordingly. These would limit results show that the hydrophobic/hydrophilic bal-
the increase in the molecular weight of the copolymers. ance can be adjusted for different applications by
Moreover, unlike the common chemical esterifying cat- varying the L-MA ratios in the monomer feed.
alyst, the molecular chain length had an enormous
effect on lipase-catalyzed polymerization. In other Thermal properties
words, the lipase reacts at different rates according to Figure 7 shows the DSC curves of the samples from
the substrate chain length.22 The enzyme has higher ac- B1 to B8. Table III summarizes the results of DSC
tivity for short- and medium-chain substrates, with less analyses. The melting point (Tm) and melting fusion
(DHf) of the polymers decreased with increasing L-
MA content in the monomer feed, and the melting
range of the polymers become broader. Beyond 60
mol % L-MA in the diacids, no melting transition
peaks appeared in polymers. This indicates that the
crystallinity of the polymer decreases with increas-
ing L-MA content in the copolymers and forms
amorphous polymers as the molar ratios of L-MA in
the diacids exceeds 60 mol %. Among the polymers,
the pure homopolymer B1 [poly(octylene adipate)]
had the highest ability to crystallize, and A8 [poly
(octylene malate)[ could not crystallize. By increas-
ing the L-MA content in the copolymers, the crystal
structure of copolymers was disrupted and the aver-
age length of the crystallizable sequence was short-
ened. All these contributed to a decrease in melting
temperature (Tm) and a broader melting range.
Accordingly, melting fusion (DHf) decreased from
Figure 7 DSC melting endotherms of the copolymers
with different L-MA content. 111.8 to 32.5 J/g with increasing molar ratio of L-MA
1120 YAO ET AL.

copolymers) in the solvents have significantly influ-

enced the lipase-catalyzed polymerization.

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