Beruflich Dokumente
Kultur Dokumente
Diana Aparaschivei1
Valentin Badea1
Carmen G. Boeriu2
Francisc Peter1
Romania; Carmen G. Boeriu, Wageningen Food & Biobased Research, P.O. Box 17, 6700 AA
†
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
[10.1002/biot.201700629].
1
Abstract
Developments of past years placed the bio-based polyesters as competitive substitutes for
renewable sources, e.g. 10-hydroxystearic acid, 12-hydroxystearic acid, ricinoleic acid, and
well as a native lipase. MALDI-TOF-MS and 2D-NMR analysis confirmed the formation of
linear/branched and cyclic oligomers with average molecular weight around 1200 and
polymerization degree up to 15. The appropriate selection of the biocatalyst and reaction
temperature allowed the tailoring of the non-cyclic/cyclic copolymer ratio and increase of
the total copolymer content in the reaction product above 80%. The catalytic efficiency of
the best performing biocatalyst (Lipozyme TL) was evaluated during 4 reaction cycles,
showing excellent operational stability. The thermal stability of the reaction products was
assessed based on TG and DSC analysis. This new synthetic route for biobased oligomers
with novel functionalities and properties could have promising biomedical applications.
hydroxyhexadecanoic acid; RCA, ricinoleic acid; PDI, polydispersity index; Dpmax, maximum
polymerization degree; NC, non-cyclic copolymer; CC, cyclic copolymer; Mn(C), number
average molecular weight of the copolymer; Mw(C), weight average molecular weight of the
copolymer.
2
1 Introduction
The bio-based polymer synthesis has emerged as a topic of interest, due to the real demand
for bio-plastic products. Among this arsenal of valuable materials polyesters are the most
promising, based on their unique and various properties suitable for development of novel
Poly(ε-caprolactone) (PCL) was one of the major synthetic biocompatible polymers, used in
some application in the biomedical field until other materials with superior properties
the development of tissue engineering gained revived interest for PCL in the last period, due
to its good biocompatibility and superior rheologic and viscoelastic properties compared to
other aliphatic polyesters [4]. Moreover, recent findings seem to ensure a privileged
position for caprolactone-based polymers in the forthcoming era of biobased products and
technologies. PCL and its monomer ε-caprolactone (ECL) own physical and chemical
properties allowing the design of a wide range of tunable biomaterials and innovative
polymer architectures [5]. Recently, Schmidt et al. reported an enzyme cascade synthesis of
ε-caprolactone and its oligomers from cyclohexanone [6]. As cyclohexanone can be easily
synthesized from phenol, resulted by the pyrolysis of lignin, this finding can open the route
for biobased PCL. Although PCL is a valuable biomaterial with important applications for
drug-delivery systems [7] and bioresorbable scaffolds [8], several drawbacks such as low
hydrophilicity, low melting point (~60°C), and relatively slow biodegradability impeded its
extensive development [4]. To overcome these drawbacks, basically two solutions are
available: blending with other biomaterials, like hydrophilic polymers [9] or synthesis of
copolymers with improved properties [10]. In the same time, melting point in the
physiological range can be advantageous for specific biomedical applications, while slow
degradation rate results in longer stability of the implants [5]. Therefore, the optimal
3
properties of these polymers depend mainly on the specific requirements of the targeted
application.
Vegetable oils are a high-class renewable source for synthesis of various polymers,
were suitable for specialty and commodity applications [13,14]. Notwithstanding of these
chemical polymerization. Lipases have been framed as powerful tool for polyester
catalyzed by lipases also opens the route for the manufacture of fully biobased polymers
obtained from renewable resources [21], but also for specialty products like aromatic-
aliphatic oligoesters [22] and functional aliphatic oligoesters [23]. The lipase from Candida
antarctica B demonstrated special ability to catalyze the synthesis of linear and star
oligomers. Some drawbacks of the traditional chemical methods related to traces of metal
catalysts, toxicity and high reaction temperatures can be avoided using the biocatalytic
route [24]. Our group reported the lipase-catalyzed synthesis of several estolides, oligomers
of hydroxy-fatty acids derived from vegetable oils, in organic reaction media [25]. Apart of
lipase, cutinase, another lipolytic enzyme, was also found able to catalyze the
homopolymerization of ω-hydroxy acids, with different chain specificity than Novozym 435
[26]. Recent advances in this field confirmed that cutinases can catalyze the synthesis of
4
The copolymers of fatty acids and hydroxy-fatty acids have promising applications as drug
[28]. Despite the well-known advantages of biocatalysis for the manufacture of innovative
copolyesters are scarce. Random copolyesters were obtained from vegetable oil derivatives,
diphenylether, at temperatures up to 90C, using Novozym 435 lipase [29]. The enzyme-
catalyzed copolymerization of ECL with hydroxy-fatty acids was not yet reported, but it may
to both homopolymer products, PCL and estolides. The chemical synthesis of these
copolymers was recently accomplished by our group [30], but the main interest is to
may result in copolymers with lower molecular mass compared to the chemical catalysis,
but the well-known selectivity of the enzymes could allow better control of the synthesized
compounds structure. In the present work, four structurally different hydroxy-fatty acids
2.1 Materials
lipase from Pseudomonas fluorescens (Amano AK), were purchased from Sigma Aldrich.
5
Novozym® 435 (lipase from Candida antarctica B immobilized on acrylic resin) and
compressible silica gel carrier), were products of Novozymes (Denmark). Two CLEA
Pseudomonas stutzeri lipases, with general applicability (CLEA P. stutzeri) and designated
for organic media (CLEA P. st. OM), were obtained from CLEA Technologies (Delft, The
Netherlands). The organic solvents (toluene, tetrahydrofuran) were purchased from Merck
and their water content (determined by Karl Fischer titration) was below 0.01%.
laboratory by using cell-free extract of E. coli TOP10 cells containing the plasmid pBAD-
HISA-OH. The recombinant cells containing the oleate hydratase and expression were
performed as previously reported [31]. The oleic acid conversion reaction was carried out
24 h at 30C and 200 rpm in 50mM TRIS-HCl buffer, pH 8 by using 0.5 ml cell free extract
Samples were taken at designated time intervals and a derivatization assay was performed
prior to the HPLC analysis, as described elsewhere [31]. Separation was performed by using
a C18-RP column (4.6x50 Merck ChromolithSpeedROD), with binary gradient elution (A:
H2O with 0.1% v/v trifluoroacetic acid, B: ACN) at 50°C, 1ml min-1 flow rate and UV detection
at 242 nm. The gradient was started with 50% B for 3 min, 50-80% B 2 min, and isocratic
elution (80% B) over 7 min. When a full conversion was indicated by the HPLC results, the
pH was adjusted to 2.0 with HCl, the mixture was filtered through Whatman filter paper and
the white solid precipitate was dissolved in acetone afterwards. The insoluble residues
were removed by filtration, and the acetone was evaporated by using rotary evaporation.
The product was dried overnight at 40C and stored at -20C until use. Structural
characterization of the synthesized 10HSA was accomplished by GC-MS, FT-IR and NMR
(see supporting information). The m/z peaks at 215, 229 and 331 in the mass spectrum
6
resulted from the GC-MS analysis (Fig. 1S, supporting information) are in accordance with
the values reported before for the trimethylsilyl derivative of 10HSA [32].
Fourier Transform Infrared (FT-IR) spectra were obtained in attenuated total reflectance
equipped with a Platinium ATR, Bruker Diamond Type A225/Q. Spectra were collected in
the range 4000-400 cm-1 with a resolution of 4 cm-1 and with 64 co-added scans.
10HSA NMR: 1H (400 MHz, pyridine-D5) δ(ppm): 3.89-3.83 (m, 1H, CHOH), 2.51 (t, J=7.3Hz,
2H, CH2CO), 1.68-1.51 (m, 28H), 0.85 (t, J=7.2Hz, 3H, CH3);
CH2,15-CH2), 29.9 (4-CH2), 26.8 (8-CH2,12-CH2), 26.0 (3-CH2), 23.3 (17-CH2), 14.7 (CH3)
The syntheses were performed by adding the lipase, about 50 U/mmol substrate, to a molar
ratio of 1:1 ECL:HFA (30-100 mM), brought to a final volume of 2 ml, using toluene as
been used, with circular mixing movement to ensure an efficient mass transfer, without
grinding the support particles. The reactions have been carried out at temperatures
between 45°C and 85°C, under continuous stirring at 350 rpm for 24 h. At the end of the
reaction the enzyme was removed by centrifugation (13,000 rpm, 3 min)/filtration, and the
MALDI-TOF MS analysis was carried out for composition analysis of polymers, using an
acceleration voltage of 25 kV, using DCTB as matrix and KTFA as ionization agent. The
7
sample preparation and analysis were performed as previously described [22]. The number
average molecular weight Mn(C), weight average molecular weight Mw(C) of the copolymers
(non-cyclic and cyclic), as well as the polydispersity index PDI have been calculated as
GC-MS analysis was performed using a Thermo Analytic GC-MS system composed from
Trace 1310 gas chromatograph, MS module and TriPlus RSH autosampler. Thermo
Scientific TG-5ms 30 m x 0.25 mm x 0.25 µm capillary column was employed in the following
conditions: oven temperature 100–300⁰C with 10⁰C min-1 heating rate, injector
temperature 300⁰C, carrier gas (helium) flow 1.0 mL min-1. The HFA conversion/formation
was determined after derivatization with BSTFA+TMCS (99:1), at 2:1 reagent: sample ratio
(w/w), for 1h at 95C, as previously described [31]. Hexadecane was used as internal
standard.
NMR spectra of the isolated product were recorded on a Bruker AVANCE III spectrometer
operating at 500.0 MHz (1H) and 125.0 MHz (13C). The samples were dissolved in
tetrahydrofurane-d8 and the chemical shifts δ are given in ppm, relative to TMS.
The thermogravimetric analysis of the oligoesters were performed by using TG 209 F1 Libra
To the best of our knowledge, this is the first report about the biocatalytic copolymerization
of ECL and HFAs. Three of the selected HFAs, specifically 16HHDA, 12HSA and RCA, have
been used in our previous study [25] as substrates for the synthesis of estolides
8
(homopolymers of HFA) catalyzed by lipases, but not for copolymers. Although the
enzymatic synthesis of 10-hydroxystearic acid (10HSA), starting from oleic acid and oleate
hydratase from different sources was reported by several groups [35, 36], 10HSA based
polyester synthesis and characterization was not yet accomplished. Since 10HSA is not
FT-IR, GC-MS, 1H-NMR, and 13C-NMR (see supporting information). The purity of the
obtained 10HSA was higher than 95% and it was used for the synthesis of copolymers with
The copolymerization reactions were carried out adding the hydroxy-fatty acid (10HSA,
16HHDA, 12HSA, or RCA) to ECL dissolved in toluene and were started by the addition of a
lipase, at different temperatures in the range 40-80°C, for 24 h. At the end of the reaction,
the enzyme was removed by centrifugation, the product was dried at 25°C and analyzed by
FT-IR, MALDI-TOF MS, and NMR techniques, to identify the formed compounds. The
reaction time of 24h was selected based on preliminary experiments (data not presented),
showing that in this time interval an almost complete conversion of ECL (the more reactive
monomer) and high conversion of HFA were achieved. Toluene has been used as reaction
medium in all experiments, due to the acceptable solubility of all substrates in this solvent.
The insertion of hydroxy-fatty acids unit into the PCL backbone was demonstrated by
and non-cyclic reaction products, chain length and dispersity, based on the similarity
between the calculated and identified molecular masses of the different oligomeric products
present in the reaction mixture. The identified copolymers were linear (in the case of
16HDDA), branched (in the case of 10HSA, 12HSA and RCA), and cyclic oligoesters with
different number of HFA units in the backbone. Figure 1a and 1b present two typical MALDI-
9
(copolymers of 12HSA_ECL and RCA_ECL, respectively), displaying the peaks corresponding
to the appropriate oligoesters. As example, the m/z values of 1050.1, 1278.5 and 1392.8
represent the K+ adducts of the (RCA)n-(ECL)m copolymer products with inserted RCA units,
where n=3 and m=3-5. The m/z values 1116.1, 1290.4 and 1458.9 demonstrate the
where n=1-2 and m=6-8. In accordance with our previous results concerning the chemical
synthesis of ECL copolymers with HFA [30], the formation of both homopolymers, PCL and
estolides, was also detected (m/z values of 1121, 1235, 941, 1216 in Figure 1). The reaction
products identified by MALDI-TOF MS are depicted in Figure 2. Even if the synthesis of PCL
and estolides as secondary reaction products could not be avoided, their formation in the
lowest possible amount was one of the objectives of this work, capitalizing the expected
The biocatalyst with the best catalytic efficiency for the synthesis of the targeted
Novozym 435 (from Candida antarctica B), Lipozyme TL (from Thermomyces lanuginosus),
as well as two CLEA Pseudomonas stutzeri lipases (one specific for organic media and one
with general applicability). Additionally, the native lipase from Pseudomonas fluorescens
was also tested, based on our previous study which proved it as the best-performing
biocatalyst for estolides synthesis [25]. The conversions were monitored by GC-MS analysis,
using hexadecane as internal standard. After 24h reaction time, the ε-caprolactone (ECL)
conversions were higher than 98%, regardless of the HFA co-substrate used, due to the
higher reactivity of the 7-member cycle lactone monomer compared to the hydroxy acids.
Consequently, the obtained copolymers contained more inserted ECL units, forming the
backbone of the products. Equimolar ratio of monomers was used in all experiments, to
10
keep the PCL copolymer amount in the reaction mixture as low as possible and to obtain
The results presented in Table 1 show that higher HFA co-monomer conversions and higher
average molecular weights were achieved when 16HHDA was used as co-substrate. Among
the tested lipases, Novozym 435 and Lipozyme TL were the most efficient for all selected
substrates, while the CLEA biocatalysts led to lower molecular weight products. Although
displaying good catalytic activity in the polyesterification reaction, the native lipase from
Pseudomonas fluorescens was less efficient than the best immobilized lipases. The
composition of the reaction mixture was estimated based on the MALDI-TOF data, as
selectivity for the formation of non-cyclic (linear or branched) copolymers, excepting those
with RCA. The highest relative amount of non-cyclic copolymer was 83%, in the
copolymerization product with 10HSA, while the highest polymerization degree was
between 11 and 15, depending on the nature of HFA used. Despite this variance, it is obvious
that structural differences between the HFA have no essential influence on the
for a variety of HFAs and the optimal conditions can be tailored for every substrate.
Compared to the chemical synthesis of 16HHDA and RCA copolymers, when tin(II) 2-
ethylhexanoate was used as catalyst at similar ECL: HFA molar ratio and higher
temperatures [30], the biocatalytic route led to higher average molecular weights (Mw
around 1300, using Novozym 435 as catalyst) and comparable content of oligoesters with
HFA units (between 73% and 83%, depending on the enzyme and HFA investigated). Only
for the oligomers of 12HSA, the average molecular weights of the products of the
biocatalytic route were lower compared to the chemical catalysis. It is noteworthy the high
saturated hydroxy acids with secondary hydroxy groups (12HSA and 10HSA), relative to
11
the cyclic copolymer. These results prove the ability of lipases to catalyze these reactions
Particularly, the synthesis of polymers involving 10HSA was not reported before, neither by
degree was studied considering the copolymerization of 16HHDA and ECL, at 1:1 molar
ratio and 30 mM substrate concentration, in the 40-80C temperature range. The selected
biocatalyst was Lipozyme TL, due to the higher relative content of non-cyclic copolymer in
the product, compared to Novozym 435, as shown in Table 1. The results, depicted in Figure
3 indicate an increase of the average molecular weight up to 35% when the temperature
was raised in the range 40C - 80C. Behind this overall increase of the molecular weight,
the selectivity of the lipase for the non-cyclic related to the cyclic form of the copolymer
decreased at higher temperature (Table 1S, supporting information), while the relative
content of copolymer reached more than 80% at 80C. The increase of the polymerization
degree at higher temperature is normal, although the reaction is also influenced by the
thermostability of the enzyme. Noteworthy, the influence of the temperature on the non-
cyclic/cyclic polymer ratio is also of special interest, since an important goal of the
product composition than obtained by chemical catalysis. Considering this influence and
the selectivity of the lipase, a tailored non-cyclic/cyclic copolymer ratio can be designed,
For a possible further increase of the average molecular weight of the products, a second
temperature study has been carried out in two steps. In this regard, the reactions were
performed first at 80C for 24h, then the temperature has been increased at 120C for
additional 24h. The aim was to check whether the increase of the temperature beyond the
12
inactivation limit of the enzyme could favor the chain elongation of the copolymer. The
results showed an increase of not more than 15% of the average molecular weight and
about 10% of the relative content of the total copolymer in the reaction product (Table 2S,
supporting information). It can be concluded that the thermal inactivation of the Lipozyme
MALDI-TOF MS and NMR spectroscopy were used for the structural characterization of the
reaction products. It should be pointed out that the products were not fractionated;
therefore, signals assigned to the homopolymers were present in the spectra, as well.
However, the insertion of hydroxy-fatty acid moieties into the hydrophobic PCL backbone
could be proved by 2D NMR spectra 1H-13C HMBC. In a typical example (Figure 6S-8S,
supporting information), showing the 16HHDA_ECL reaction product, the distance coupling
of the carbon atom from the carbonyl moiety from 173.7 ppm (C-1) and the protons from
4.18 ppm (H2C-33, 3J), 2.4 ppm (H2C-2, 2J) and with the protons from 1.69 ppm (H2C-3,
3J).
The 13C NMR spectra revealed the presence of several signals in the region specific for
carbonyl groups which indicate the presence of the several products species, as the MALDI-
The thermal stability of the co-polyesters was evaluated by TG analysis (Figure 4),
compared with the homopolymers (PCL and the appropriate estolides). Unlike PCL, the
profiles of the reaction product and the correspondent estolide showed multistep
degradation processes. The first degradation step (up to 300C) corresponds to the loss of
small oligomers and the results confirm that the 16HDDA_ECL samples contain higher
molecular weight polyesters. The overlaid TG curves (Figure 2 and Figures 9S-14S in
supporting information) clearly indicate that the HSA-ECL copolyesters start to degrade at
13
lower temperatures compared to PCL. The same behavior was observed when poly(ε-
stability of the copolymers was higher compared to the estolides, as 35% mass was lost up
to 300C, compared to 45% registered for the 10HSA and 12HSA estolides. Compared to the
PCL homopolymer, the thermal stability of the copolymers was lower, excepting the
Contrary to the parent homopolymer (PCL), the TGA curve of the copolymer clearly shows
The melting temperature (Tm), the melting enthalpy (Hm) and degree of crystallinity were
determined by DSC measurements in the 20-590C range, at 10Kmin-1 heating rate. The
degree of crystallinity was calculated through the relation between Hm of each sample and
the theoretical value of a 100% crystalline PCL sample [38]. As a general tendency, the
copolyesters showed higher melting temperature values compared to PCL (58.9C), but
lower than the corresponding estolides. Among the four new synthesized materials, the
highest Tm values were registered for the 16HHDA_ECL product (Table 3S, supporting
observed when 10HSA and 12HSA were used as co-monomer, compared to the value
calculated for 16HHDA based products. These results are in concordance with the
successive reaction cycles without important loss of activity and consequently reducing the
consecutive reaction cycles for the synthesis of the 16HHDA_ECL copolymer, at 80C. This
study was carried out at higher temperature to explore the behavior of the biocatalyst at
prolonged utilization in more adverse conditions (according to the producer, the optimum
14
temperature of this enzyme is in the range of 50-75C). The reaction products were
increase of the calculated average molecular weights during the biocatalyst reuse. The
increase of the polymerization degree during the reuse of the biocatalyst is positive and
demonstrates the high operational stability of the biocatalyst, although it was not significant
The relative copolymer content was higher than 80% throughout all reaction cycles,
although an increase of the relative content of cyclic copolymers to the detriment of the
linear copolymers was observed after each reuse. This observation is in accordance with
the increase of the relative amount of cyclic copolymer at higher temperature, as discussed
earlier. Such an increase of the cyclic/non-cyclic copolymer ratio during the repeated use of
a biocatalyst at the same temperature was not yet reported and currently there is no
4. Conclusion
Linear, branched, and cyclic oligoesters were successfully obtained for the first time from
different biobased HFAs and ECL, using a biocatalytic route. The synthesis of copolymers
of 10HSA is of special interest, considering that this renewable monomer was obtained from
oleic acid by a biocatalytic process, as well. Among the tested biocatalysts, the commercially
available lipases from Candida antarctica B (Novozym 435) and Thermomyces lanuginosus
(Lipozyme TL) were the most efficient, leading to higher average molecular weight and
copolymer content of the synthesized products. Thermal analysis revealed superior thermal
stability of the reaction products compared to the correspondent estolides. The structural
analysis of the copolymers by MALDI-TOF MS and NMR demonstrated that several HFA
units were inserted into the polymer backbone. The sequence of ECL and HFA units in the
polymer chains looks random and any control of the insertion mechanism was not yet
possible. However, we proved that the biocatalytic process allows the synthesis of these
15
oligoesters with higher selectivity compared to the chemical route. Particularly, a new
> 88% HFA conversion and > 80% branched copolymer content in the reaction product.
Moreover, considering the very plausible manufacture of ECL from biobased resources in
the near future, the present process can open the way for an entirely biobased route for ECL
copolymers with new functionalities, providing very attractive applications in the field of
double bond of the RCA_ECL copolymer, followed by the insertion of a specific functional
group, could be another important future research direction. Our studies will be focused on
Acknowledgement
This work was supported by a grant of the Romanian Authority for Scientific Research and
PNCDI III. The authors are grateful to Prof. Isabel Arends and Dr. Linda Otten (University of
Delft) for supplying the oleate hydratase from Elizabethkingia meningoseptica, used for the
Conflict of interest
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Table 1.
The ability of various lipases to catalyze the synthesis of oligoesters of ECL and hydroxy-
19
Table 2.
The efficiency of the Lipozyme TL lipase in multiple polyesterification reaction cycles of ECL
and 16HHDA, in toluene, at 80C. The length of each reaction cycle was 24 h.
20
Figure legends
Figure 2. Reaction scheme for the synthesis of copolyesters from hydroxy-fatty acids and
ε-caprolactone, emphasizing the products obtained with 10-HSA. Dotted curves signify the
Yellow triangle symbols represent the values of the total copolymer content in the reaction
mixture
Figure 4. The TG (A) and DSC (B) curves of the 16HHDA_ECL copolyester (brown),
21
Figure 1a
22
Figure 1b
Figure 2
23
Figure 3
Figure 4a
24
Figure 4b
25