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Cite this: DOI: 10.1039/c5gc00992h

Received 9th May 2015,
Accepted 12th June 2015
DOI: 10.1039/c5gc00992h
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Enzymatic reactive extrusion: moving towards

continuous enzyme-catalysed polyester
polymerisation and processing
S. Spinella,a,b,c M. Ganesh,a G. Lo Re,c S. Zhang,c J.-M. Raquez,c P. Duboisc and
R. A. Gross*a

This paper demonstrated the feasibility of conducting an enzymatic ring-opening polymerisation by reactive extrusion (REX) at
high shear and temperature conditions. Using immobilized
Candida antarctica Lipase B (CALB) as catalyst at temperatures
ranging from 90 to 130 C, -pentadecalactone (PDL) was converted (>99%) by REX at 60 RPM for 15 min to PPDL with Mw
163 000 g mol1.

The majority of current polymerisation methods use metal

catalysts. Residual metal catalysts are often undesirable in
materials used for biomedical and electronic applications.1
Immobilized enzyme-catalysts have been shown to have distinct advantages relative to most metal catalysts including: (1)
naturally derived, (2) low toxicity, (3) high chemo- and regioselectivity, (4) activity at relatively low temperatures and (5) no
need for strict exclusion of water and oxygen.26 The most commonly employed lipase for enzyme-catalysed ring opening
polymerisations (eROP) and polycondensations is the immobilized lipase form of Candida antarctica Lipase B (CALB). Of the
many lactonic substrates for which CALB is an active polymerisation catalyst, CALB eciently catalyses ROPs of larger
lactones (e.g. -pentadecalactone, PDL).3 The immobilized
CALB catalyst used in ref. 4 and herein is Novozyme 435
(N435). ROP of larger lactones are known to be dicult for

a
Department of Chemistry and Biology, Center for Biotechnology and
Interdisciplinary Studies, Rensselaer Polytechnic Institute (RPI), 4005B
BioTechnology Building, 110 Eighth Street, Troy, New York 12180, USA.
E-mail: grossr@rpi.edu
b
Department of Chemical and Biomolecular Engineering, NYU Polytechnic School of
Engineering, Six Metrotech Center, Brooklyn, New York 11201, USA
c
Centre dInnovation et de Recherche en MAtriaux Polymres CIRMAP, Service des
Matriaux Polymres et Composites, University of Mons, Place du Parc 23, B-7000
Mons, Belgium
Electronic supplementary information (ESI) available: MR spectra of Poly(w-pentadecalactone prepared by e-REX (10% N435, 90 C), molecular weight
time course data as a function of reaction time; linear regression analysis of the
eect of N435 concentration; tabulated data from granulometry; tabulated
residual enzyme activity data. See DOI: 10.1039/c5gc00992h
Current address: SyntheZyme LLC, 11 University Place, Rensselaer NY, 12144,
USA.

This journal is The Royal Society of Chemistry 2015

many organometallic catalysts because the polymerisations are

primarily entropy-driven.7 Nevertheless, recent progress has
resulted in a number of chemical catalysts that successfully
convert PDL to high molecular weight polymers. Examples of
these catalysts are aluminium salen and terdentate phonoxyimineamine aluminium. Problems encountered with these
catalysts are as follows: (i) synthesis from expensive ligands,
(ii) requiring inert reaction conditions (e.g. performed in a
glove box) and (iii) the use of solvents.8,9
The ability of lipases to catalyse ring-opening and condensation polymerisations at relatively low temperatures (e.g.
7090 C) is advantageous to reduce energy input and to preserve thermally sensitive chemical moieties. However, when
high molecular weight polymer synthesis is desired, corresponding diusional constraints must be overcome by either
running reactions at higher temperatures (e.g. 150220 C),
which is generally regarded as not feasible for enzyme-catalysts
that denature under such conditions,10 or by adding solvent.
For example, for N435-catalyzed synthesis of high molecular
weight (Mn > 50 000 g mol1) poly(-pentadecalactone), PPDL,
and poly(-caprolactone, PCL), the viscosity was lowered by the
addition of toluene. Subsequently, the final polymer products
are obtained by precipitation into a non-solvent such as
methanol.1113 Solvent-based processes reduce the volumetric
productivity of reactions and also require solvent recycling.
Reactive extrusion (REX) is an industrially relevant technique because it combines polymerisation and processing into
a single step.14 REX has been used to overcome the aforementioned problems of bulk polymerisations, mainly reaction
mixing and heat transfer, that slow chain growth. In one
example, poly(-caprolactone) (PCL) with Mn 200 000 g mol1
was produced by REX using aluminium triisopropoxide as the
catalyst with only a 5 minute residence time.15 Enzymes have
been used in twin-screw extruders for food-related applications
(i.e. depolymerisations).1618 However, to the best of our knowledge, enzymatic polymerisations have not been performed
using REX.
The objective of this study was to determine whether
enzyme activity for lipase-catalysed polymerisations can be

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maintained at the high temperatures, confined bulk conditions and mechanical shearing created by the co-rotating
screws and for times required by REX to reach high molecular
weight polyesters. The possibility of directly extruding the
polymer out of the die was also examined. Initial e-REX experiments were conducted at 90 C with 10% N435 based on optimized conditions used for batch solution (toluene)
polymerisations.19 An initial mixing speed of 60 RPM was
chosen based on previous research.20
Fig. 1 shows the force produced by screw rotation as a function of REX temperature and N435 loading. The viscosity of
the polymer is directly related to extruder force. In another
words, more viscous products requiring more energy to maintain the same mixing speed.22 The force versus reaction
time graph shown in Fig. 1 has three distinct regions:
(1) initial decrease in the force, (2) a plateau and, finally, (3) a
sharp increase in the viscosity of the product. This can be
explained by that, during the initial stages of the reaction, the
monomer is melting, leading to an overall decrease in the viscosity of the system. Subsequently, in the plateau region,
enzymesubstrate complexes form that initiate chain growth.

Fig. 1 Force as a function of reaction time for reactive extrusion polymerisations of PDL catalyzed by 2.5, 5 and 10% N435 and performed at
90 C and 110 C.

Finally, the polymerisation progresses such that high

monomer conversion and large increases in polymer molecular weight occur, resulting in high viscosity reaction media.
PPDL prepared at 90 C with 10% N435 reached sucient
chain length (see below) after 20 min of polymerisation in the
extruder, and began to crystallize, thus resulting in high
force.23 The increase in viscosity for N435-catalyzed polymerisations performed by REX is similar to chemical catalysed REX
polymerisations.14,22
PPDL, recovered after 20 min (90 C, 10% N435) from the
extruder, was analysed directly by SEC and NMR (without fractionation, e.g. by precipitation). Its Mn is 90 000 g mol1 ( =
2.0) and PDL conversion reached >99% (Table 1). This is in
contrast to PDL polymerisations performed in bulk for 24 h
with 20% N435, which gave PPDL with comparatively low Mn
(15 300 g mol1) and higher dispersity ( = 4.4). Furthermore,
increasing the reaction time to 72 h for a bulk reaction
resulted in a small increase in Mn from 15 300 to 22 100 g
mol1 (Table 1).8 Larger values for aforementioned reactions
conducted in bulk are attributed to the formation of oligomers
which, due to diusion constraints, do not condense with
other oligomers to form longer chains.5,21 Polymerisations performed in the extruder at 90 C for 15 min gave similar molecular weights as well as values similar to those performed in
toluene at 85 C for 72 hours (Table 1, entry 4 vs. entry 1).
Moreover, proton NMR analyses (shown in Fig. SI-1) showed
greater than 99% conversion of PDL after 15 minutes of extrusion. The relatively short reaction time required to reach high
Mn PPDL by e-REX relative to batch solution and bulk N435catalyzed PPDL reactions is attributed to the confined bulk
conditions and mechanical shearing created by the co-rotating
screw and the ability of the immobilized enzyme to retain
activity under such these conditions.
In the previous example, reactions were conducted at
90 C, which is below PPDLs peak melting transition (Tm) at
97 C and close to the crystallization temperature (85 C).23
To explore the possibility of continuous manufacturing by
reactive eROP extrusion, where the polymer is extruded from
the die after >99% monomer conversion, and to determine the
feasibility that REX may be performed with shorter residence
times, the eect of temperature on the enzymatic reactive
extrusion was explored.

Table 1 Comparison of polymerisations to prepare PPDL that were conducted using enzymatic reactive extrusion (e-REX) and batch polymerisations in bulk and in solution

Entry

Polymerisation

Time (h)

Temp (C)

N435a (%)

Mnb (g mol1)

b,c

Conversione (%)

1
2
3
4

e-REX
Bulk21
Bulk21
Solution,11,d

0.25
24
72
72

90
80
80
85

10
20
20
10

90 000
15 300
22 100
80 000

2.0
4.4
3.0
2.0

>99
>99
>99
>99

Novozyme 435, physically immobilized Candida antarctica Lipase B (CALB) immobilized on Lewatit. b Molecular weights were determined by
size exclusion chromatography (SEC) with chloroform as the eluent using narrow dispersity polystyrene standards. c = Mw/Mn. d Reaction
performed in toluene at 0.15 mol PDL (33.7 g) in 29.6 mL toluene. e Determined by 1H NMR, signals corresponding to residual monomer were
not observed.

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Table 2 Eect of temperature on the reactive extrusion polymerisation

of PDL catalyzed by 10% N435 performed at dierent temperatures with
the reaction time and mixing rate xed at 15 min and 60 RPM,
respectivelya

Temp (C)

Mnb (g mol1)

90
95
100
105
110
120
130

86 000
83 000
84 000
84 000
78 000
80 000
81 500

1.9
2.0
2.2
2.2
1.9
2.1
2.0

Conversions are >99% from 1H NMR analysis.

were determined by SEC.

Molecular weights

The temperature was varied from 90 to 130 C with the residence time and mixing rate fixed at 15 min and 60 RPM,
respectively (Table 2). In all cases, complete PDL conversion
was observed. The reaction temperature did not have a significant eect on PPDL Mn, which ranged from 86 000 to 78 000.
Complete monomer conversion at these high temperatures
infers that, as the polymerisation proceeds, the enzyme maintains sucient residual activity for complete monomer conversion. This retention of activity for N435 even at 130 C is
important since this infers that residence times can be shortened and REX polymerisations using N435 can be conducted
for higher melting (e.g. 110 C) polyesters.24 Indeed, based on
the time required to reach 3500 N, the reaction time decreased
from 15 to 10 min by conducting e-REX at 90 and 130 C,
respectively.
Control experiments were performed without the addition
of N435 at both 90 C and 110 C for 60 min, and no conversion of PDL to PPDL was observed by 1H NMR. This verifies
that N435 is active in the extruder and the polymerisation is
not autocatalysed by high shear stresses generated during the
extrusion process. By performing the reaction above PPDLs
Tm, the final product was extruded out of the die. Experiments
were also performed to determine the eect of N435 concentration on PDL conversion and PPDL molecular weight. A processing temperature of 110 C was chosen since this allows the
material to be extruded out of the die at the end of the polymerisation. As the %-by-weight N435 relative to monomer was
decreased from 10 to 5 and 2.5%, the time required to reach
an axial force of 6000 N increased in a manner that follows a
linear relationship (r2 = 0.967, Fig. SI-2). A study of
%-monomer conversion and molecular weight change versus
time was performed for polymerisations conducted by eREX at
110 C with 2.5% N435. The results plotted in Fig. 2 and listed
in Table SI-1 show that, initial chain growth is slow (Mn =
1600 at 10 min), and is attributed to an apparent lag period for
at least the first 10 min. Origins of the initial lag time are
attributed to the time required for enzyme-monomer activation
and chain initiation to occur. Indeed, Kobayashi reported that
chain initiation is the limiting step in e-ROP.25 After this
initial lag time, the Mn increases linearly with PDL %-conver-

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Fig. 2 Relationship between molecular weight and monomer conversion for the reactive extrusion polymerisation of PDL catalyzed by 2.5%
N435 at 110 C with a mixing rate of 60 RPM.

sion to 95% (Fig. 2). Thereafter, a large increase in Mn from

121 000 to 142 000 g mol1 occurs with a relatively small
increase in monomer conversion (about 95 to >99%). This is
consistent with previous reports and corresponds to a change
from chain-growth to a step-growth mechanism as polycondensation between chain ends becomes increasingly competitive
with lactone ring-opening from chain ends.26 Furthermore,
the general characteristics of this e-REX polymerisation of PDL
conducted at 110 C with 2.5% N435 agrees well with the
mechanism proposed for the N435-catalyzed batch solution
polymerisation of -caprolactone, CL, to form PCL, in toluene
at 60 C.26
N435 beads were recovered after e-REX (10%-N435, 90 C,
15 min, mixing rate of 60 RPM) by dissolving the reaction
mixture in xylene and separation of the enzyme beads by filtration. Prior to use, the N435 beads are spherical with no
visible surface defects (Fig. 3A). Optical microscopy of recovered beads from e-REX shows little change in bead integrity
but that physical abrasion occurs at bead surfaces (Fig. 3B).
Potential changes in bead dimensions due to the e-REX
were evaluated by granulometry.27 Measurements were
recorded for the mean particle size (D 50, m) and from both
extremes of the particle size window (D 10 and D 90, m).

Fig. 3 Optical microscopy pictures of: (A) an unused N435 bead and (B)
a bead recovered after 90 C for 15 minutes at 60 RPM.

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The results listed in Table SI-2 show that a decrease in the

bead size after e-REX is observed but the change in size is
small.
In addition to study of changes in bead physical characteristics, recovered N435 beads after e-REX (10%-N435, 90 C,
15 min, mixing rate of 60 RPM) were analysed to determine
their residual activity. The assay used is described in detail
elsewhere.28 In summary, the unused and recovered N435
beads were assayed to determine monomer conversion by
1
H-NMR for -caprolactone polymerization conducted in
toluene-d8 at 80 C. Results from this experiment are listed in
Table SI-3. Comparing the slopes of CL conversion versus
time, the recovered enzyme beads have 25% lower activity. To
gain further information on the recovered beads, the protein
content was determined and compared to unused N435. This
experiment is important since N435 is manufactured by physical adsorption of CALB onto and within macroporous poly
(methyl methacrylate), PMMA, beads. Presumably, if enzyme
leaching occurs, the detached free enzyme migrates into the
surrounding polymer product. Since the bead material is constructed from PMMA, nitrogen content in N435 is directly proportional to the bead protein content. X-ray photoelectron
spectroscopy (XPS) was performed on unused and recovered
beads, %-nitrogen values were converted to %-protein content
in beads and the results are listed in Table SI-4. The control
(unused) beads have 8.4 1% CALB whereas N435 recovered
after e-REX has 4.5 0.5% or 54% of the protein in unused
beads. The migration of enzyme from the beads into product
does not imply that the leached enzyme becomes inactive;
however, free CALB activity should dier from immobilized
CALB. The extent of this change in enzyme activity is currently
unknown.
Since the recovered beads have a lower CALB content, the
dierence in activity between the unused and recovered
enzyme beads may be due to enzyme leaching and not enzyme
denaturation during e-REX. Previous work by our group has
shown that loses in protein due to enzyme-leaching are not
proportional to loses in bead activity.29 Furthermore, the
%-enzyme lost can greatly exceed the observed activity loss of
the recovered enzyme beads.29

Conclusions
This paper explored the feasibility of performing lipase-catalysed polyester synthesis by REX at temperatures up to 130 C,
under confined bulk conditions and with mechanical shearing
created by the co-rotating screws. Surprisingly, the immobilized enzyme withstood these conditions. The ecient mixing
by REX shortens reaction times, decreases catalyst requirements and enables the formation of high molecular weight
polyester under bulk reaction conditions. Furthermore,
immobilized lipase e-REX reached high monomer conversions
and polymer molecular weights such that the synthesized
polymer was extruded without the need for post-reaction
product purification (e.g. by precipitation). In contrast, N435-

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catalyzed PDL polymerisations conducted in bulk using a

batch reactor did not exceed Mn values of about 20 000 g mol1
due to diusion constraints. For reaction temperatures up to
130 C (15 min, 60 RPM), N345 retained sucient activity such
that high PDL conversion and PPDL molecular weight (>99%
and Mn 81 500) was achieved. The ability to conduct e-REX
polymerisations up to 130 C suggests that shorter residence
times, substantially below 15 min, may be sucient to achieve
high molecular weight polyesters. Furthermore, higher
melting polymers (e.g. about 110 C, 20 C below the extrusion
temperature) may also be synthesised and directly extruded by
this method. Future work will build on the findings disclosed
herein to develop practical processes in which the residence
time is decreased to a few minutes and the enzyme is immobilized within the extruder such that stronger interactions
between the support and the enzyme prohibit enzyme leaching. These improvements will facilitate continuous processes
in which the immobilized enzyme can be re-used over multiple
cycles. Methods of shortening reaction time currently under
investigation include using higher activity lipases and cutinases from other sources or that are improved by protein
engineering.30,31 Furthermore, improvements in the catalyst
will be needed that increase is kinetic and thermal stability.

Acknowledgements
The authors thank the National Science Foundation Partnership for Innovation (NSF-PFI) program (Award #1114990)
entitled Next Generation Bioplastic Nanocomposites for
financial support. Financial support from Wallonia and European Commission in the frame of SINOPLISS-POLYEST project
and OPTI2MAT program of excellence and from FNRS-FRFC
are also gratefully acknowledged.

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