Bisepoxide cross-linked enzyme aggregates - New immobilized biocatalysts for selective biotransformations

Article abstract of DOI:10.1002/cctc.201300806

Glycerol diglycidyl ether (GDE) is a convenient and inexpensive bisepoxide cross-linker as demonstrated by the preparation of cross-linked enzyme aggregates (CLEAs) from two enzyme classes. The GDE CLEAs of lipase from Pseudomonas fluorescens (AK), lipase from Burkholderia cepacia (PS), and lipase B from Candida antarctica (CaL B) as well as of phenylalanine ammonia-lyase (PAL) from Petroselinum crispum demonstrated improved properties as compared with their glutaraldehyde (GA) cross-linked counterparts. Ultrasonication studies indicated that the GDE CLEAs of lipase PS and PAL were mechanically more stable than the GA CLEAs. In the kinetic resolution of rac-1-phenylethanol, the catalytic activity of the GDE-lipase CLEAs (U=69.6, 134.8, and 127.4 U g ^-1 for AK, CaL B, and PS prepared at 22 °C, respectively) surpassed that of the corresponding GA-lipase CLEAs (U=24.4, 131.0, and 119.2 U g^-1 for AK, CaL B, and PS prepared at 22 °C, respectively). The GDE co-CLEAs from PAL and bovine serum albumin (BSA) could be recycled at least three times if used for the stereoselective ammonia addition in 6 M ammonia to (E)-3-(thiophen-2-yl)acrylic acid, whereas the recycling of the conventional GA-PAL CLEAs from this medium failed. The missing linker: Glycerol diglycidyl ether is applied as a cross-linker for cross-linked enzyme aggregates (CLEAs) of various enzymes such as lipases and phenylalanine ammonia lyases. The bisepoxide CLEAs prove to be efficient and robust biocatalysts surpassing the performance of the glutaraldehyde CLEAs.

Full text of DOI:10.1002/cctc.201300806

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DOI: 10.1002/cctc.201300806
Bisepoxide Cross-Linked Enzyme Aggregates—New
Immobilized Biocatalysts for Selective Biotransformations
[
a]
[b]
[a, c]
[a]
[d]
Diꢀna Weiser, Andrea Varga, Klaudia Kovꢀcs,
Flꢁra Nagy, Andrꢀs Szilꢀgyi,
[c, e]
[b]
[a, f]
Beꢀta G. Vꢂrtessy,
Csaba Paizs, and Lꢀszlꢁ Poppe*
Dedicated to Professor Jꢀnos Rꢁtey on the occasion of his 80th birthday.
Glycerol diglycidyl ether (GDE) is a convenient and inexpensive
bisepoxide cross-linker as demonstrated by the preparation of
cross-linked enzyme aggregates (CLEAs) from two enzyme
classes. The GDE CLEAs of lipase from Pseudomonas fluorescens
rac-1-phenylethanol, the catalytic activity of the GDE–lipase
À1
CLEAs (U=69.6, 134.8, and 127.4 Ug for AK, CaL B, and PS
prepared at 228C, respectively) surpassed that of the corre-
À1
sponding GA–lipase CLEAs (U=24.4, 131.0, and 119.2 Ug for
(
AK), lipase from Burkholderia cepacia (PS), and lipase B from
AK, CaL B, and PS prepared at 228C, respectively). The GDE co-
CLEAs from PAL and bovine serum albumin (BSA) could be re-
cycled at least three times if used for the stereoselective am-
monia addition in 6m ammonia to (E)-3-(thiophen-2-yl)acrylic
acid, whereas the recycling of the conventional GA–PAL CLEAs
from this medium failed.
Candida antarctica (CaL B) as well as of phenylalanine ammo-
nia-lyase (PAL) from Petroselinum crispum demonstrated im-
proved properties as compared with their glutaraldehyde (GA)
cross-linked counterparts. Ultrasonication studies indicated
that the GDE CLEAs of lipase PS and PAL were mechanically
more stable than the GA CLEAs. In the kinetic resolution of
Introduction
[
1,2]
The use of new biocatalytic methods is a rapidly developing
area of synthetic chemistry both on the laboratory and on the
industrial scale because biocatalysts are selective, easy to
affect the efficiency of biocatalysts.
Immobilization can
often improve these key properties as well as enable the re-
[3,4]
covery and reuse of these biocatalysts.
Various immobiliza-
[
1]
handle, and environmentally friendly. Selectivity, specificity,
catalytic activity, and enzyme stability are key factors that
tion techniques may improve enzyme properties in several
ways, such as by increasing rigidity of the enzyme, by prevent-
ing subunit dissociation through multipoint, multisubunit im-
mobilization, and/or by generating a favorable environment
[3,5]
for the enzyme.
Immobilization may also modulate diffusion
[
a] D. Weiser, K. Kovꢀcs, F. Nagy, Prof. Dr. L. Poppe
Department of Organic Chemistry and Technology
Budapest University of Technology and Economics
M u˝ egyetem rkp. 3, H-1111 Budapest (Hungary)
Fax: (+36)1-463-3697
characteristics, mechanical resistance, thermal and operational
[3,5]
stability, and other properties of the resulting biocatalysts.
Cross-linked enzyme aggregates (CLEAs) were developed as
[6,7]
simple and robust immobilization tools.
They are generally
E-mail: poppe@mail.bme.hu
[
b] A. Varga, Dr. C. Paizs
prepared through precipitation with a suitable solvent in the
absence of any carrier and then cross-linking of the aggregates
formed by a multifunctional cross-linker such as glutaralde-
Biocatalysis Research Group
Babe s¸ -Bolyai University of Cluj-Napoca
Arany Jꢀnos str. 11, Ro-400028 Cluj-Napoca (Romania)
[6,7]
hyde (GA; Scheme 1).
The advantage of CLEAs is improved
[
c] K. Kovꢀcs, Prof. Dr. B. G. Vꢁrtessy
Institute of Enzymology
Research Centre for Natural Sciences of Hungarian Academy of Sciences
Karolina fflt 29, H-1113 Budapest (Hungary)
specific and volumetric activity compared to most of the carri-
er-based immobilized enzyme preparations as well as ease of
[
6,7]
recovery and reuse.
Softness, low mechanical stability, and
[3c]
[
d] Dr. A. Szilꢀgyi
diffusion limitations are the major drawbacks of CLEAs. In
the preparation of CLEAs of various enzymes, GA is usually the
Department of Physical Chemistry and Materials Science
Budapest University of Technology and Economics
Budafoki fflt 8, H-1111 Budapest (Hungary)
[
6,7]
first reagent of choice.
However, at lower concentrations, it
may exist in the monomeric form simultaneously as the free al-
dehyde, the hydrate, or the hemiacetal. At higher concentra-
tions, GA polymerizes to oligomeric hemiacetals and all these
forms can react with proteins in different ways and lead to im-
mobilization by forming Schiff’s base bonds and several other
[
e] Prof. Dr. B. G. Vꢁrtessy
Department of Biotechnology and Food Sciences
Budapest University of Technology and Economics
Szt. Gellꢁrt tꢁr 4, H-1111 Budapest (Hungary)
[
f] Prof. Dr. L. Poppe
SynBiocat Ltd
Lꢀzꢀr deꢀk u 4/1, H-1173 Budapest (Hungary)
[8]
bond types (Scheme 1). Because the forms of GA participat-
ing in protein immobilization depend on various factors, such
as concentration, pH, temperature, and solvent composition,
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201300806.
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[22]
ammonia to (E)-cinnamic acid.
PALs of plant and yeast origin
were also useful as biocatalysts
in the preparation of various un-
natural l- and d-a-amino
[16,19,23]
acids.
Because cross-link-
ing stabilizes multimeric en-
[24]
zymes, it is not surprising that
isolated PALs were immobilized
[9]
as simple CLEAs or BSA co-
[25]
CLEAs with GA.
In addition to the factors in-
vestigated most frequently in
CLEA
choice of solvent, amount of
GA, and addition of surfactants
optimization
studies
(
[
6,7]
and other chemicals),
other
aldehyde cross-linkers, such as
dextran polyaldehyde, were
used if GA was detrimental to
Scheme 1. Preparation of CLEA biocatalysts.
[26]
enzyme activity. Moreover, the
co-immobilization of the active
enzyme with an inert protein
such as the globular, lysine-rich
the protocol for using GA must be specified in detail for each
BSA proved to be beneficial as it was demonstrated by the im-
proved properties of enzyme–BSA co-CLEAs from BSA and
[
6–8]
protein.
[27]
General methods using GA as a cross-linker are known for
many enzymes such as oxidoreductases, chloroperoxidase,
lipase B from Candida antarctica (CaL B) or from BSA and
[25]
PAL. By increasing the cross-linking density, the use of BSA
improved stability and mechanical resistance of CLEAs made of
[
6,7]
penicillin acylase, proteases, lipases,
and phenylalanine am-
[
9]
[27]
monia-lyase (PAL). Although the recyclabilities of CLEAs pre-
CaL B, with relatively few lysines exposed on the surface.
pared from various enzymes including lipase from Pseudomo-
Similarly, the positive effect of BSA was due to the greatly in-
creased number of cross-links between the larger lysine-poor
homotetrameric PAL (molecular mass 302 kDa) and the smaller
[
10]
[9]
nas sp. and PAL (in the elimination reaction from l-Phe)
have been documented, an improvement in the catalytic activ-
ity and stability of the conventional CLEAs is a timely objec-
[25]
lysine-rich monomeric BSA (molecular mass 66.5 kDa).
[
4]
tive. Therefore, we decided to study bisepoxides as a new
class of cross-linkers in the generation of CLEAs of lipases and
PALs, which represent two enzyme classes.
Poly(glycidyl ethers), such as glycerol diglycidyl ether (GDE)
and poly(ethylene glycol) diglycidyl ether, are widely used as
additives for cross-linking polymers bearing amine, hydroxyl,
or carboxyl groups. For example, polyfunctional epoxides such
as glycidyl ethers reacting with polyamines represent an im-
Lipases are monomeric enzymes (triacylglycerol hydrolases,
EC 3.1.1.3) and by far the most often used biocatalysts in sev-
[
11]
[28]
eral applications, such as in bakery, as a laundry detergent
additive, and even in alternative energy strategies to convert
portant class of epoxy hardeners. Although a study using
poly(ethylene glycol) diglycidyl ether for the preparation of mi-
[
12]
[29]
vegetable oils into biofuels. In living organisms, lipases cata-
croelectrode biosensors indicated the reactivity, usefulness,
[
13]
lyze the hydrolysis of triglycerides to fatty acids and glycerol
and biocompatibility of such compounds toward enzymes as
polyamines, their use as cross-linking agents has not been re-
ported for the preparation of CLEAs. Bisepoxides such as GDE
are useful cross-linkers in forming CLEAs owing to their ability
to form stable bonds under mild conditions not only with the
amine groups of lysine but also with the sulfur- and oxygen-
containing residues of cysteine, tyrosine, aspartate, or gluta-
mate. Furthermore, GDE is an inexpensive, partially water-solu-
ble bisepoxy compound; it is less toxic than GA.
[
14]
at the lipid–water interface. In synthetic processes, lipases
can catalyze a wide range of enantio- and regioselective reac-
tions such as hydrolysis, esterification, transesterification, ami-
[
15]
nolysis, and ammoniolysis. In other immobilization methods,
[
6,7]
CLEAs of lipases proved to be efficient biocatalysts.
PALs (EC 4.3.1.24 and EC 4.3.1.25) are homotetrameric en-
zymes catalyzing the nonoxidative deamination of l-Phe to
[
16]
(
E)-cinnamic acid. In plants, PALs are essential at the starting
[
17]
point of the phenylpropanoid pathway. The presence of PAL
To explore the value of GDE as a cross-linker for the prepara-
tion of CLEAs (Scheme 1), lipases and PALs were selected as
two classes of enzymes with important synthetic applications.
This study demonstrates, for the first time, the usefulness of
GDE as a convenient bisepoxy-type cross-linker for the prepa-
ration of CLEAs and co-CLEAs of different monomeric and mul-
[
18]
[19]
was also reported in fungi and in some bacteria. In medi-
cal applications, PAL is a potential therapeutic enzyme in
[
20]
cancer treatment or in the enzyme replacement therapy of
[
21]
phenylketonuria. PAL has synthetic potential as a biocatalyst
in the large-scale production of l-Phe through the addition of
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timeric enzymes, which provides biocatalysts with improved
characteristics.
Results and Discussion
Preparation and morphology of CLEAs from lipases and
PALs
CLEAs prepared with GDE and GA differ in several aspects
(Scheme 1). For example, the cross-link with GDE (11-heavy
atom-long chain with hydrogen bond donor and acceptor
groups) is longer and more hydrophilic than that with GA
(five-carbon-long aliphatic chain) under dilute, slightly basic
conditions. Moreover, the CÀX bonds (X=NH, S, O) forming in
GDE cross-linking, in contrast to the C=N bonds forming in GA
cross-linking, are not susceptible to hydrolysis.
Although the operational and thermal stability and recycling
of CLEAs from various enzymes were examined and document-
[
4–7]
ed,
it is important to demonstrate the mechanical stability
differences of CLEAs prepared with different cross-linkers
through the analysis of their particle size distribution after
strong ultrasonication. The particle size distribution of CLEAs
from lipase PS and PAL before and after ultrasonication (for
Figure 2. Change in the particle size distribution of PAL aggregates cross-
linked with a) GDE and b) GA after ultrasonication for 300 and 600 s.
3
00 and 600 s) is shown in Figures 1 and 2, respectively.
pending on which form of GA takes part in cross-link forma-
The GDE CLEAs demonstrated a more uniform particle size
[8]
tion. The high variability of cross-linking with GA may explain
distribution [final mean particle size ꢀ26 mm for lipase PS (Fig-
ure 1a) and ꢀ16 mm for PAL (Figure 2a)] and better resistance
to mechanical stress after ultrasonication than the correspond-
ing GA CLEAs [42 mm for lipase PS (Figure 1b) and 19 mm for
PAL (Figure 2b)]. These differences may be due to the different
nature of cross-links with GA and GDE. As mentioned earlier,
quite diverse types of cross-links can be formed with GA, de-
the less uniform particle size distribution of the GA CLEAs than
that of the corresponding GDE CLEAs. In contrast, bisepoxides
are less inclined to polymerize at nearly neutral pH values than
GA. Although the epoxide rings of GDE may react with a wider
[28]
selection of nucleophilic residues than GA (Scheme 1, X=
NH, S, and O), the topology of forming cross-links comprising
an 11-heavy atom-long chain with the monomeric GDE is more
uniform than that of the cross-links generated with GA. Be-
cause the epoxide functions of GDE may form cross-links not
only via the surface-exposed amine functions of lysine residues
but also via sulfur and oxygen atoms of cysteine, tyrosine, as-
partate, and glutamate, the cross-link density may be higher in
the GDE CLEAs than in the GA CLEAs, especially in the case of
proteins with only few surface-exposed lysine residues. These
differences may rationalize the more uniform particle size dis-
tribution as well as the higher mechanical stability of the GDE
CLEAs than that of the GA CLEAs.
A comparison of the non-cross-linked coaggregates pre-
pared from PAL and BSA and those cross-linked with GDE and
GA (co-CLEAs) by using SEM also revealed remarkable differen-
ces (Figure 3). The SEM images for the non-cross-linked coag-
gregates of PAL and BSA demonstrated an assembly of amor-
phous particles (Figures 3a and S2a) whereas the aggregates
of pure PAL contained crystal-like structures (Figure S2b). PAL
CLEAs (Figure S3a and b) and PAL–BSA co-CLEAs (Figures 3b
and c) were less structured than the non-cross-linked aggre-
gates. The GDE–PAL–BSA co-CLEAs appeared as porous struc-
tures (Figure 3b) with a larger available surface than that of
the nonporous GA–PAL–BSA co-CLEAs (Figure 3c). A higher
degree of porosity was also observed for the GDE–PAL CLEAs
(Figure S3b) compared with the GA–PAL CLEAs (Figure S3a).
Figure 1. Change in the particle size distribution of lipase PS aggregates
cross-linked with a) GDE and b) GA after ultrasonication for 300 and 600 s.
I=relative intensity, d=diameter.
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Lipase CLEAs and the corresponding precipitated but non-
cross-linked aggregates were prepared from three frequently
used lipases: AK (from Pseudomonas fluorescens), PS, and
CaL B. The lipase aggregates were tested as enantiomer-selec-
tive biocatalysts in the kinetic resolution of rac-1-phenyletha-
nol (rac-1) with vinyl acetate in an organic medium (Scheme 2
and Table 1). We were pleased to find that as a new cross-
Table 1. Catalytic properties of various lipase CLEAs in the kinetic resolu-
[
a]
tion of rac-1 with vinyl acetate.
[
b]
Lipase
Cross-linker
T
[
U
[Ug
C
[%]
ee(R)-2
E
À1
8C]
]
[%]
[
c]
AK
AK
AK
AK
–
4
4
22
4
22
4
4
22
4
22
4
4
22
4
6.2
75.9
69.6
6.6
24.4
3.2
60.7
134.8
10.9
131.0
11.6
30.6
127.4
70.7
119.2
2.2
97.9
99.2
99.4
98.6
99.2
97.2
99.8
99.7
99.2
99.7
99.3
99.6
99.4
99.5
99.4
100
GDE
GDE
GA
GA
[
27.5
25.3
2.4
8.9
1.2
22.0
48.9
3.9
47.5
4.2
11.1
46.2
25.7
43.2
>100
>200
>200
>200
72
@200
@200
>200
@200
>200
@200
@200
@200
@200
AK
c]
CaL B
CaL B
CaL B
CaL B
CaL B
PS
PS
PS
PS
PS
–
GDE
GDE
GA
GA
[
c]
–
GDE
GDE
GA
GA
22
[
a] Reaction conditions: hexane/tert-butyl methyl ether 2:1, t=2 h, T=
308C; [b] Temperature of precipitation from acetone and cross-linking;
[
c] Precipitated aggregates without cross-linking.
linker, GDE proved to be quite efficient. GDE was an effective
cross-linker for CLEAs from all the three lipases (Table 1).
Notably, cross-linking is sensitive to temperature.
Figure 3. SEM images of A) coprecipitated PAL and BSA and PAL–BSA coag-
gregates cross-linked with B) GDE and C) GA.
As expected, cross-linking by oxirane ring opening with the
reactive groups of the protein exposed on the surface was in
all cases more pronounced at 228C than at 48C. Cross-linking
with GA demonstrated similar behavior because all GA–lipase
CLEAs prepared at 228C performed better than those prepared
at 48C. The activity difference between GDE and GA CLEAs
was larger if precipitation and cross-linking were performed at
48C. In the case of lipases (AK and CaL B), the activity of GDE
CLEAs prepared at 48C was 6–10 times higher than that of the
GA CLEAs prepared at 48C. It can be assumed that at a low
temperature, mainly Schiff’s base-type bonds were formed
from the monomeric GA, which resulted in a lower degree of
cross-linking and multipoint stabilization. Even at this low tem-
perature, epoxides may react not only with amines but also
with S- and O-nucleophiles, which leads to denser cross-link-
Stereoselective biotransformations with lipase CLEAs
To correlate morphology data of various CLEAs of lipases and
PALs with their catalytic performances, as a typical example,
a stereoselective reaction was studied for each type of enzyme
(Scheme 2).
[8]
ing. At a higher temperature, various oligomeric forms of GA
can enhance cross-linking but with GDE an even denser cross-
link network may be formed. Notably, with all three lipases
(
AK, PS, and CaL B), the best GDE CLEAs (prepared at 228C)
proved to be superior to their GA counterparts in terms of
both activity (see U and c in Table 1) and enantiomer selectivity
(
see ee(
and E in Table 1). These results—at least the im-
R)À2
Scheme 2. Biotransformations with lipase and PAL biocatalysts.
proved activity—can be due to the differences in cross-link
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density and the fact that the cross-linker in GDE CLEAs is
longer, which results in a looser structure with less limitation
of diffusion.
It has already been shown that even the mechanically less
stable GA CLEAs were recyclable (e.g., lipase PS CLEAs could
be recycled 10 times from a kinetic resolution in an organic
[
10]
medium with only 19.1% loss of activity ). Therefore, the re-
cycling of lipase CLEAs was not studied herein.
Stereoselective biotransformations with PAL CLEAs
Various CLEAs were prepared from pure recombinant PAL
(from P. crispum) and tested in ammonia elimination from
l-Phe, their natural substrate (data not shown). It was demon-
strated earlier with the nonimmobilized pure PAL that the reac-
tion from rac-2-amino-3-(thiophen-2-yl)propionic acid (rac-3;
with a tiophen-2-yl moiety) is a kinetic resolution and elimina-
tion from 3 proceeds faster than the elimination from the nat-
[
23d]
ural substrate l-Phe.
P. crispum) with either GA or GDE as cross-linkers were tested
for ammonia elimination from this fast-reacting substrate 3
Therefore, CLEAs from pure PAL
(
(Scheme 2).
The GA–PAL CLEAs (prepared from the crude supernatant of
Rhodotorula glutinis fermentation) were reported to be recycla-
ble up to 12 times in the production of l-Phe through ammo-
[
9]
nia addition to (E)-cinnamate. In addition, the GA–PAL–BSA
[
29]
co-CLEAs increased the thermal stability of CaL B and crude
[
25]
PAL. Because a previous study with the native form of pure
PAL (from P. crispum) showed that ammonia addition to ((E)-3-
(
thiophen-2-yl)acrylic acid) (4) resulted in the stereospecific for-
[
23d]
mation of (S)-3,
the behavior of PAL–BSA co-CLEAs (from
Figure 4. Recycling of PAL–BSA coaggregates cross-linked with GDE and GA
in A) ammonia elimination from rac-3 and B) ammonia addition to 4.
P. crispum) was investigated further in the elimination reaction
from 3 and the addition reaction onto 4 (Scheme 2 and
Figure 4).
Although both GA and GDE–PAL CLEAs were active as bio-
catalysts in the elimination reactions from l-Phe or rac-3, none
of them were active in the reverse reaction (i.e., ammonia ad-
dition to the acrylate 4; data not shown). In contrast, the GA
and GDE–PAL–BSA co-CLEAs were active both in the ammonia
elimination from 3 (Figure 4a) and in the ammonia addition to
Our results indicated that GDE cross-linking increases not only
the mechanical stability of the CLEAs formed but also the op-
erational stability compared with those of the GA CLEAs.
4
(Figure 4b). These results may be rationalized by assuming Conclusions
a tight packing of CLEAs from pure PAL, which may involve
severe limitation of diffusion. Owing to the different sizes of
BSA and PAL, the PAL–BSA co-CLEAs may have a looser struc-
ture, which results in less limitation of diffusion and less loss of
activity on recycling. This assumption may be in accordance
with the activity of the GA–crude PAL CLEAs (from R. glutinis)
Glycerol diglycidyl ether (GDE) was used as a new and efficient
bisepoxide-type cross-linker for the cross-linked enzyme aggre-
gates (CLEAs) of various enzymes such as lipases and phenyl-
alanine ammonia-lyases.
The GDE CLEAs were efficient and robust biocatalysts sur-
passing the performance of the corresponding glutaraldehyde
CLEAs. The use of GDE and similar bisepoxide cross-linkers
widen the scope of the cross-linking technology for enzyme
immobilization.
[
9]
in ammonia addition to (E)-cinnamate because proteins in
the crude supernatant other than PAL may cause a similar “di-
lution effect” as the BSA in our PAL–BSA co-CLEAs.
Recycling studies revealed remarkable stability differences
between GA and GDE–PAL–BSA co-CLEAs (Figure 4). Although
the GDE–PAL–BSA co-CLEAs retained a significant portion of
their initial activities after the third reaction cycle even in the
alkaline medium used for ammonia addition to 4 (6m ammo-
nia solution, pH 10.0; Figure 4b), the GA–PAL–BSA co-CLEAs
were found to be completely inactivated after the first cycle.
Experimental Section
The sources of materials and enzymes and details for analyses are
provided in the Supporting Information.
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Preparation of lipase and PAL CLEAs
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and ethanol for PAL or acetone for lipase (3 mL, anhydrous) as
a precipitant were mixed and shaken at RT for 10 min. The GA or
GDE solution (80 mL, 2%) was added to the mixture with continu-
ous shaking (450 rpm). After 24 h, the mixture was centrifuged
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[
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pH 8.8, followed by centrifugation: 2200 g, 15 min, 88C) and once
with ethanol (suspension in 10 mL, followed by centrifugation:
2
200 g, 15 min, 88C). Before use, CLEAs were kept in ethanol
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Acknowledgements
The authors acknowledge the financial support of the Hungarian
OTKA Foundation (NN-103242) and the New Szꢁchenyi Develop-
ment Plan (TMOP-4.2.1/B-09/1/KMR-2010-0002: “Development
of quality-oriented and harmonized R+D+I strategy and func-
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the Romanian National Authority for Scientific Research, CNCS-
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support of COST Action CM1303 (SysBiocat). We thank Prof.
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aggregate · enzyme catalysis · immobilization · lipase ·
phenylalanine ammonia-lyase
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