Microscopic monitoring provides information on structure

Transcription

Biotechnology
Journal
Biotechnol. J. 2014, 9
DOI 10.1002/biot.201300049
www.biotechnology-journal.com
Research Article
Microscopic monitoring provides information on structure
and properties during biocatalyst immobilization
Sarka Bidmanova1,2, Eva Hrdlickova3, Josef Jaros4, Ladislav Ilkovics4, Ales Hampl2,4, Jiri Damborsky1,2,3
and Zbynek Prokop1,2,3
1 Loschmidt
Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment
RECETOX, Faculty of Science, Masaryk University, Brno, Czech Republic
2 International Clinical Research Center, St. Anne’s University Hospital Brno, Czech Republic
3 Enantis Ltd., Brno, Czech Republic
4 Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
Enzymes have a wide range of applications in different industries owing to their high specificity
and efficiency. Immobilization is often used to improve biocatalyst properties, operational stability, and reusability. However, changes in the structure of biocatalysts during immobilization and
under process conditions are still largely uncertain. Here, three microscopy techniques – brightfield, confocal and electron microscopy – were applied to determine the distribution and structure
of an immobilized biocatalyst. Free enzyme (haloalkane dehalogenase), cross-linked enzyme
aggregates (CLEAs) and CLEAs entrapped in polyvinyl alcohol lenses (lentikats) were used as
model systems. Electron microscopy revealed that sonicated CLEAs underwent morphological
changes that strongly correlated with increased catalytic activity compared to less structured, nontreated CLEAs. Confocal microscopy confirmed that loading of the biocatalyst was not the only factor affecting the catalytic activity of the lentikats. Confocal microscopy also showed a significant
reduction in the pore size of lentikats exposed to 25% tetrahydrofuran and 50% dioxane. Narrow
pores appeared to provide protection to CLEAs from the detrimental action of cosolvents, which
significantly correlated with higher activity of CLEAs compared to free enzyme. The results showed
that microscopy can provide valuable information about the structure and properties of a biocatalyst during immobilization and under process conditions.
Received
Revised
Accepted
Accepted
article online
29 JAN 2014
31 JAN 2014
14 MAR 2014
17 MAR 2014
Supporting information
available online
Keywords: Cross-linked enzyme aggregates · Immobilization · Microscopy · Polyvinyl alcohol · Structure
1 Introduction
Correspondence: Assoc. Prof. Zbynek Prokop, Loschmidt Laboratories,
Department of Experimental Biology, Faculty of Science, Masaryk
University, Kamenice 5/A13, 625 00 Brno, Czech Republic
E-mail: [email protected]
Additional Correspondence: Prof. Jiri Damborsky, Loschmidt Laboratories,
Department of Experimental Biology, Faculty of Science, Masaryk
University, Kamenice 5/A13, 625 00 Brno, Czech Republic
E-mail: [email protected]
Abbreviations: CLEAs, cross-linked enzyme aggregates; lentikats, lensshaped particles of polyvinyl alcohol
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Recent developments of biotransformation processes
have been driven by the increasing demand for sustainable, energy-saving, and environmentally friendly synthetic strategies [1, 2]. Although enzymes are biocatalysts
with high efficiency and excellent selectivity, their widespread application in industrial processes is often limited
due to poor reusability and low operational stability [3].
Many immobilization strategies have been developed to
improve biocatalyst properties [4–7]. Maximum retention
of the enzymatic activity has been the primary concern,
whereas less attention has been paid to the biocatalyst
structure and its changes during immobilization and use
under process conditions. This is surprising considering
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that the number and size of immobilized biocatalysts, distribution of the biocatalyst particles in the immobilization
matrix, properties of matrix, e.g., particle size and porosity, are all expected to have a crucial effect on the final
activity [8, 9]. Further, the selection of the most suitable
immobilization method for the intended application
under given conditions, e.g., in organic solvents or at
increased temperature, is usually carried out by empirical
screening [10]. Rational identification is mostly impossible, as the microscopic structure and molecular basis of
enzyme immobilization have not yet been fully elucidated
[11]. Structural analysis can simplify this process by
enabling preliminary evaluation of the selected immobilization method or matrix under given conditions.
Previous studies on the relationship between the
structure and catalytic properties of immobilized biocatalysts have mostly investigated carrier-free immobilization by employing bright-field and electron microscopy
[12–16]. Immobilization within a matrix limits the number
of techniques that can be applied. However, fluorescence
confocal microscopy has been employed to give a threedimensional visualization of a biocatalyst’s distribution
within an immobilization matrix and provide an understanding of the diffusional restrictions [17].
In the present study, three different microscopy techniques, i.e., bright-field, confocal and electron microscopy,
were used in combination to systematically investigate
the structural behavior of an enzyme during immobilization and under typical process conditions. Haloalkane
dehalogenase LinB from Sphingobium japonicum UT26
[18] was selected as a model enzyme owing to its wide
application potential, e.g., in biocatalysis, bioremediation,
decontamination of warfare agents, biosensing and protein tagging [19]. This enzyme was immobilized in crosslinked enzyme aggregates (CLEAs) [20] and its mechanical properties were further enhanced by entrapment into
lens-shaped particles of polyvinyl alcohol (lentikats) [21].
Comparison of structural microscopic data with catalytic
activity data revealed important information about the
underlying structure–activity relationships.
2 Materials and methods
2.1 Materials
Recombinant histidine-tagged haloalkane dehalogenase
LinB from S. japonicum UT26 [18] was produced in Escherichia coli BL21(DE3) and purified as described previously
[22]. Prepared LinB was lyophilized using an ALPHA 1-2
LD freeze dryer (Martin Christ, Germany). Ammonium ferric sulfate, N,N-dimethylformamide, polyethylene glycol
with a molecular weight of 600, 1000, 4000, or 6000 and
potassium dihydrogen phosphate were all purchased
from Fluka (Switzerland). Ampicillin sodium salt and isopropyl-β-D-thiogalactopyranoside were obtained from
2
Duchefa (The Netherlands). Nitric acid was purchased
from Lach-Ner (Czech Republic). Polyvinyl alcohol was
kindly provided by LentiKat’s (Czech Republic). Acetone,
diethyl ether, ethanol, isopropanol and methanol were
purchased from Chromservis (Czech Republic). All other
chemicals were purchased from Sigma-Aldrich (USA). All
reagents used were of analytical grade.
2.2 Enzyme immobilization
2.2.1 Preparation of dehalogenase CLEAs
Haloalkane dehalogenase LinB (38.4 mg), lyophilized in
50 mM phosphate buffer, and bovine serum albumin
(38.4 mg) were dissolved in 9.6 mL of water in a centrifuge
tube. Dissolved proteins were precipitated by adding
28.8 mL of saturated ammonium sulfate (pH 8.0) and
allowing it to react for 45 min under stirring. Next, the
solution was mixed with 3.1 mL of dextran polyaldehyde
[23] and stirred for a further 45 min. The precipitation and
cross-linking steps were performed in an ice bath. After
cross-linking, the suspension was subjected to centrifugation at 4000 g for 20 min at 4°C. The supernatant, containing residual ammonium sulfate and free enzyme, was
subsequently withdrawn and the resulting CLEAs were
resuspended in 30.7 mL of saturated sodium hydrogencarbonate. Sodium borohydride (61.4 mg) was added to
the solution and the mixture was allowed to react for
30 min at 4°C under stirring. Dehalogenase CLEAs were
washed three times with 50 mM phosphate buffer (pH 7.5)
and separated by centrifugation at 4000 g for 10 min at
4°C. Optimization of the dehalogenase CLEAs is described
in detail in the Supplementary Material.
2.2.2 Preparation of dehalogenase lentikats
Immobilization of the dehalogenase CLEAs into lentikats
was performed as described previously [24]. Polyvinyl
alcohol and polyethylene glycol were heated in distilled
water until the polyvinyl alcohol particles had dissolved
completely. The mixture was then cooled to 35°C. A dispersion of dehalogenase CLEAs (0.5, 1.0, or 1.5 g) was
slowly added to the polymeric mixture to give a final
CLEA content of 10, 20, or 30 wt%, respectively. After
thorough mixing, small droplets of the resulting mixture
were dripped onto a smooth plastic plate, which was then
dried at 30°C. After drying, the resulting lenses were
soaked in 0.1 M sodium sulfate for 40 min to re-swell. Prepared lentikats were washed with 400 mL of 50 mM phosphate buffer (pH 7.5) and stored at 4°C [21].
2.3 Activity assay
The enzymatic activity of free and immobilized dehalogenase LinB was determined in 10 mL of 100 mM glycine
buffer (pH 8.6) at 37°C by colorimetry [25]. Unless otherwise stated, 1,2-dibromoethane was added as substrate
to a final concentration of 8.7 mM, determined using a
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Trace GC gas chromatograph (ThermoQuest Scientific,
UK) equipped with a DB-FFAP capillary column (30 m ×
0.25 mm × 0.25 μm, Phenomenex, USA) and a flame-ionization detector. The temperature program began with an
isothermal period at 40°C for 1 min, after which the temperature was increased to 170°C at rate of 20°C per min.
The reaction was initiated by addition of dehalogenase to
the reaction mixture and terminated by mixing with 35%
v/v nitric acid. The concentration of reaction products
(bromide ions) was monitored spectrophotometrically at
460 nm using a Sunrise spectrophotometer (Tecan,
Switzerland) following addition of mercuric thiocyanate
and ferric ammonium sulfate. The concentration of reaction products was determined from a calibration curve
prepared using sodium bromide as a standard solution.
Characterization of the dehalogenase lentikats is described
in detail in the Supporting Information.
2.4 Microscopic analysis
2.4.1 Bright-field optical microscopy
Bright-field optical microscopy was used to study CLEAs
dispersed in 50 mM phosphate buffer (pH 7.5) in a ratio of
1:1 (v/v). The CLEA dispersion was spread over a glass
slide and visualized using an Olympus BX50 microscope
(Olympus, Japan) equipped with a 10× objective lens.
Images were captured using an Olympus E-410 digital
camera (Japan) mounted on the microscope. A size analysis of CLEAs was performed on 1000 particles of each type
in three replicate measurements using the software
ImageTool 3.0 (Department of Dental Diagnostic Science,
University of Texas, USA). CLEAs were classified into
different distribution groups according to their Feret’s
diameter.
2.4.3 Confocal microscopy
The preparation of samples of polyvinyl alcohol particles
with and without CLEAs for confocal microscopy followed
different staining procedures.
The polyvinyl alcohol matrix of lentikats incubated in
phosphate buffer or organic solvents was visualized using
the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic
acid. The dye (5.2 mg) was first dissolved in 20 mL of
50 mM phosphate buffer (pH 7.5) and then lentikats were
added. After 30 min incubation under shaking at 22°C,
the lentikats were washed with 200 mL of 50 mM phosphate buffer (pH 7.5). The prepared samples were stored
in the same buffer prior to microscopy analysis.
The CLEAs entrapped in lentikats were labelled using
fluorescein 5(6)-isothiocyanate (Invitrogen, USA) dissolved in dimethyl sulfoxide (final concentration of
10 mg/mL). The fluorescent dye (40 μL) was added to
20 mL of carbonate/bicarbonate buffer (15 mM sodium
carbonate, 30 mM sodium bicarbonate, pH 9.5) together
with lentikats. Staining was performed for 60 min under
shaking at 22°C. Subsequently, the lentikats were washed
with 50 mL of 0.05 M Tris sulfate buffer (pH 8.2) and frozen
in tissue freezing medium Jung (Leica Microsystems,
Germany) at −26°C using a Cryocut 1800 cryostat (Leica
Microsystems, Germany). The frozen samples were sectioned vertically and horizontally with thickness of 5 μm
using the microtome incorporated in the cryostat, then
mounted on glass slides with mounting medium Mowiol
40-88 (Sigma-Aldrich, USA). Both samples of particles
were examined using an Olympus Fluoview 500 confocal
microscope (Olympus, Japan) equipped with 10×, 20×,
and 40× objectives. Images were digitized and analyzed
using the software Fluoview (Olympus, Japan) and
ImageTool 3.0 (Department of Dental Diagnostic Science,
University of Texas, USA).
2.4.2 Scanning electron microscopy
Scanning electron microscopy was performed on both
CLEAs dispersed in deionized water and CLEAs entrapped
in a polymeric matrix. The aqueous CLEA dispersion was
spread over a glass slide (diameter of 10 mm) and dried at
22°C overnight. The CLEAs entrapped in lentikats were
fixed in 3% v/v phosphate buffered glutaraldehyde for
20 min at 22°C. The lentikats were washed three times
with 50 mM phosphate buffer (pH 7.5) for 10 min and then
postfixed in 1% v/v osmium tetraoxide for 30 min at 22°C.
Lentikats were washed a further three times with 50 mM
phosphate buffer (pH 7.5) for 10 min and then dehydrated
with different grades of ethanol (30, 50, 70, and 100% v/v,
10 min each step). The dehydrated lentikats were dried
overnight in air at 22°C.
Both samples of CLEAs were sputter coated with
gold for 3 min using an SCD040 system (Baltec, Liechtenstein) and current of 30 mA. Electron microscopy
images were obtained with a Vega TS 5136 XM scanning electron microscope (Tescan, Czech Republic)
operated at 20 kV.
3 Results and discussion
3.1 Dehalogenase CLEAs
Dehalogenase CLEAs were successfully prepared by
co-aggregation of haloalkane dehalogenase LinB and
bovine serum albumin, followed by cross-linking with a
mild cross-linker, dextran polyaldehyde, and final centrifugation. The activity retention of CLEAs prepared in
this first trial was 35.5 ± 3.6% [26]. The effects of different
precipitants, concentrations of cross-linker and addition
of protective ligand were tested and optimized (Fig. 1).
Ammonium sulfate at concentrations higher than 70%
and 3.1 mL of dextran polyaldehyde were found to be the
most appropriate reagents for preparation of the dehalogenase CLEAs (see Supporting Information, Fig. S1–S3 ).
The size distribution of the optimized CLEAs separated
by centrifugation was determined by bright-field microscopy (Fig. 2). The dispersion of dehalogenase CLEAs
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Figure 1. Optimization of dehalogenase CLEAs. (A) Effect of precipitants
on the retention of enzymatic activity of haloalkane dehalogenase LinB.
Recovery of the highest activity was observed at the concentration indicated in brackets (full datasets are shown in Supporting Information,
Fig. S1–S3). (B) Effect of varying the amount of dextran polyaldehyde
on the activity of dehalogenase CLEAs. The enzymatic activity towards
8.7 mM 1,2-dibromoethane was measured in 100 mM glycine buffer
(pH 8.6) at 37°C. The error bars show the standard deviation of three
replicate measurements (A–B). Abbreviations: DMF – N,N-dimethylformamide, DMSO – dimethyl sulfoxide, DPA – dextran polyaldehyde,
PEG – polyethylene glycol.
comprised amorphous particles with a large variation in
size. Some particles formed clusters that were easily visible to the naked eye. Most of the CLEAs had diameters of
less than 100 μm, with the highest proportion being from
0 to 25 μm (approximately 40%). Only 5% of particles were
larger than 150 μm, with a maximal observed size of
930 μm. A predominance of small-sized CLEAs is desirable for biocatalysis owing to their high surface-to-volume ratio, which facilitates the diffusion of substrate molecules to the catalytic sites [27].
The technique used for final separation of the prepared CLEAs was expected to have a significant effect on
the particle size and subsequent catalytic activity. Thus,
the effects of both centrifugation and decantation were
investigated by a combination of bright-field microscopy
and activity measurements. The size distribution of
CLEAs separated by centrifugation and decantation
showed dissimilar trends (Fig. 2) as demonstrated by the
4
significantly different mean particle size for the two
approaches: 49.5 ± 8.6 and 134.5 ± 28.4 μm for centrifuged
and decanted CLEAs, respectively (median particle size
of 30.2 ± 2.7 and 88.7 ± 29.9 μm, respectively). Whereas
the majority of CLEAs separated by centrifugation exhibited particle sizes of <25 μm, decantation resulted in partial loss of these small-sized particles, which can be attributed to their resistance in moving through the liquid as
given by Stokes’ law [28]. The highest proportion of
decanted CLEAs (approximately 27%) had particle sizes
ranging between 25 and 50 μm. In addition, the number
of larger CLEAs with sizes from 150 to 1000 μm was significantly higher (up to 17%) for the decanted compared
to centrifuged CLEAs. Unexpectedly, CLEAs obtained by
centrifugation had comparable activities to those generated by decantation (26.7 ± 3.2% and 29.4 ± 1.7%, respectively; Fig. 3), suggesting that the particle size was not the
only determinant of catalytic activity. Therefore, scanning
electron microscopy was used to obtain detailed images
of any morphological differences between the CLEAs separated by the two methods (Fig. 3). The structure of
CLEAs separated by centrifugation was significantly
affected by the centrifugation force. The particles were
clearly less structured compared to more porous CLEAs
separated under the influence of gravitational force during decantation. The particle-size distribution and morphological differences observed for the decanted CLEAs
suggest that two factors are important: (i) reduction of the
enzymatic activity due to diffusional limitations of bulkier particles; and (ii) increased enzymatic activity due to
the higher specific surface area of the decanted CLEAs.
The balance between these two effects determines the
resulting activity of the decanted CLEAs.
Because the activity of the decanted and centrifuged
CLEAs was similar, only centrifugation was used to prepare CLEAs in the subsequent experiments for practical
reasons, primarily the shorter separation time. The partial
loss of enzymatic activity as a result of morphological
changes of CLEAs during centrifugation has been shown
to be recovered by using vortexing and the FastPrep cell
disruption system [13, 29]. In the present study, we tested whether treatment of centrifuged CLEAs by sonication
would change the particle-size distribution and/or morphology of CLEAs, and thus increase their catalytic activity. Bright-field microscopy revealed similar particle-size
distributions for the centrifuged and sonicated CLEAs
(Fig. 2), with mean particle size of 49.5 ± 8.6 and
57.3 ± 14.7 μm, respectively (median particle size of
30.2 ± 2.7 and 37.2 ± 7.5 μm, respectively). Several significant morphological changes that occurred during sonication of CLEAs were clearly evident in the scanning
electron micrographs (Fig. 3). The aggregates additionally processed by sonication were more structured and less
compressed compared to the unsonicated CLEAs. The
large surface area and many cavities of the sonicated
CLEAS would allow substrate molecules to readily access
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Figure 2. Effect of separation and disruption on particle size of dehalogenase
CLEAs. (A) The size distribution was
determined using image analysis of
1000 particles of each type in three replicate measurements. (B) LogD transformation of data from A. The frequency in
graphs (A) and (B) differs due to the different box sizes used. Images of (C) centrifuged CLEAs, (D) centrifuged CLEAs
further disrupted by sonication and
(E) decanted CLEAs were acquired by
bright-field optical microscopy.
Figure 3. Effect of separation and
disruption on (A) the activity and
(B–E) structure of dehalogenase CLEAs.
(A) The enzymatic activity towards
8.7 mM 1,2-dibromoethane was measured in 100 mM glycine buffer (pH 8.6)
at 37°C. The error bars show the standard deviation of three replicate measurements. (B) The pore area was determined using analysis of minimally
700 pores in the images of (C) centrifuged CLEAs, (D) centrifuged CLEAs
further disrupted by sonication and
(E) decanted CLEAs acquired by scanning electron microscopy with a magnification of 20 000×.
the enzyme active site, whereas enzyme molecules in the
interior of the less porous centrifuged CLEAs would have
a lower probability of reacting with the substrate molecules, resulting in lower catalytic activity. The structural
changes observed by electron microscopy strongly corre-
lated with activity measurements: the sonicated CLEAs
exhibited a significantly higher activity (41.3 ± 3.0%) than
the centrifuged CLEAs (26.7 ± 3.2%, Fig. 3). These results
are in agreement with observations by Garcia-Garcia et
al. [13] and Montoro-Garcia et al. [29].
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3.2 Dehalogenase lentikats
A highly concentrated heterogeneous mixture of dehalogenase CLEAs was immobilized in a polyvinyl alcohol
matrix to generate dehalogenase lentikats. To the naked
eye, the resulting lentikats were white, elastic, lensshaped particles of diameter 3–5 mm. Entrapment of optimized CLEAs into the polymer was accompanied by a
slight decrease in the enzymatic activity (29.5 ± 2.0%)
[26]. Therefore, confocal and scanning electron microscopy (Fig. 4) were used to evaluate the factors affecting
the catalytic properties of the immobilized enzyme, e.g.,
internal structure of the matrix defined by the matrix
homogeneity, pore size distribution, mode of pores, CLEA
dispersion and loading. Microscopic analysis of microtomed horizontal sections through the lentikats revealed
that the polyvinyl alcohol had a heterogeneous structure,
with significant differences between the center and edge
of particles. Whereas the pore-size distribution at the
edge was from 0 to 10 μm (mean of 4.5 ± 1.5 μm), pores
from 4 to 28 μm (mean of 12.5 ± 4.1 μm) were predominantly observed in the center of the lenses (Fig. 5). The
pores were spherical with an area of 72.3 μm2 and a cir-
cularity of 0.9 (a value of 1.0 indicates a perfect circle,
Table 1). The interconnection of pores was clearly visible
in both the confocal and electron micrographs. This is
important to facilitate the flow of substrate to CLEAs
located in the inner structure of the lens-shaped particles.
Microscopy of microtomed vertical sections through the
lentikats showed that pores were densely distributed over
the whole scanned area except for the upper surface of the
lenses, where a separate zone without pores was formed.
Assessment of CLEAs in lentikats (Fig. 4) revealed broad
distributions for abundance (133.4 ± 101.0 aggregates per
mm–2 of matrix) and area fraction (6.9 ± 4.5%), suggesting
a non-uniform distribution of CLEAs through the lenses
(Supporting Information, Table S1). Investigation of the
size of the entrapped CLEAs showed that there were significant differences between aggregates dispersed in the
polyvinyl alcohol matrix. The size of aggregates influenced their incorporation into porous matrix: CLEAs significantly larger than the pores appeared to protrude
through a couple of pores (Fig. 4), whereas small-sized
CLEAs were partially embedded in the polymeric matrix
and located in single pores. The mean CLEA size
(16.6 ± 36.6 μm) was in agreement with the measured par-
Figure 4. (A–C) Scanning electron
microscopy images of dehalogenase
CLEAs entrapped in a polyvinyl alcohol
(PVA) matrix at a magnification of
(A) 2000×; (B) 10 000× and (C) 20 000×.
All images were acquired in the center of
a lentikat. (D–H) Effect of dehalogenase
CLEA loading on the activity and structure of lentikats. (D) The enzymatic
activity towards 8.7 mM 1,2-dibromoethane was measured in 100 mM
glycine buffer (pH 8.6) at 37°C with
intact and sliced particles. The error bars
show the standard deviation of three
replicate measurements. (E) The abundance of CLEAs was determined from
analysis of five confocal microscopy
images showing the distribution of
(F) 0.5 g, (G) 1.0 g, and (H) 1.5 g
dehalogenase CLEAs in lentikats. The
images were acquired in the center of
the lens-shaped particles at a magnification of 100×.
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Table 1. Pore parameters of lentikats incubated in phosphate buffer, dioxane or tetrahydrofuran for 1 weeka)
Pore properties
Area (μm2)
Feret’s diameter (μm)b)
Circularityc)
72.3
12.0
0.9
98.5 ± 81.6
12.8 ± 4.9
0.8 ± 0.2
50%
dioxane
25%
dioxane
Phosphate buffer
(pH 7.5)
73.1
12.9
0.8
71.4 ± 56.0
12.8 ± 3.0
0.6 ± 0.4
21.2
6.9
0.9
21.6 ± 12.1
7.0 ± 1.4
0.7 ± 0.3
25%
tetrahydrofuran
59.5
10.7
0.9
62.2 ± 37.3
10.6 ± 2.6
0.8 ± 0.2
a) Median (bold) and mean ± standard deviation were determined from confocal micrographs by image analysis of 200 pores in the center of the lentikats.
b) Feret’s diameter – the longest distance between any two points along the selection boundary.
c) Circularity – shape descriptor: 4n*area/perimeter2. A value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated shape.
ticle-size distribution of dispersed CLEAs used for the
preparation of lentikats (Fig. 2), suggesting that the
entrapment was efficient with no or negligible leaching of
aggregates. The observed microscopic structure of
entrapped CLEAs, particularly their porosity, was similar
to that observed for dispersed CLEAs treated by sonication (Fig. 3). The findings regarding the size and structure
of immobilized CLEAs were also consistent with their
measured enzymatic activity, which was only slightly
diminished compared to that of dispersed CLEAs.
The results indicated that the enzymatic activity of
the lentikats could be improved by optimizing the CLEA
loading. In particular, the microscopic inspection of the
CLEA distribution in the lentikats suggested that the
loading capacity of the polyvinyl alcohol matrix might be
even higher than used here. Therefore, the effect of CLEA
loading on the activity of lentikats was tested whilst
simultaneously assessing the quality of immobilization by
confocal microscopy (Fig. 4). The abundance and area
fraction of the CLEAs determined by image analysis were
proportional to the amount of CLEAs used for the preparation of the lentikats (Supporting Information, Table S1).
The loading had an obvious influence on the activity of
the lentikats. However, the differences were rather small
and not proportional to the content of CLEAs (Fig. 4). The
highest enzyme loading resulted in the highest activity,
but probably not all the loaded CLEAs remained active.
Too low loading of CLEAs resulted in inefficient utilization
of the polymeric matrix. However, the access of substrate
molecules to individual CLEAs was simpler than in more
loaded lentikats. To investigate the effect of diffusional
limitations on the activity of differently loaded lentikats,
the lenses were sliced and their activity was determined
(Fig. 4). Even though the activity was slightly increased
for all types of lentikats, the enzymatic activity still did not
directly correlate with the extent of enzyme loading,
which is similar to observations by Ursoiu et al. for immobilized lipase [30]. Further experiments were performed
with lentikats containing 1 g of CLEAs (biocatalyst represents 20 wt% of the lens), following the recommendation
of Schlieker and Vorlop [31].
The individual properties of the matrix and enzyme
are not the only determinants of activity of immobilized
biocatalysts [9]. The availability of a substrate is governed
by its concentration and solubility, which can be
improved by addition of an organic cosolvent. In the present study, hydrophobic bromocyclohexane was used as a
model substrate for haloalkane dehalogenase. The use of
10% tetrahydrofuran and 40% dioxane improved the solubility of the substrate by approximately 4-fold compared
to aqueous buffer (Supporting Information, Fig. S6). Further increasing the solvent concentration did not increase
the concentration of dissolved substrate.
The suitability of the polyvinyl alcohol matrix for application in organic solvents was studied by confocal
microscopy and compared with the behavior in aqueous
solution (Fig. 5, Table 1). Lentikats stored in phosphate
buffer were elastic lens-shaped particles with spherical
pores. The pore area and diameter exhibited broad distributions with mean values of 98.5 ± 81.6 and 12.8 ± 4.8 μm,
respectively (Fig. 5, Table 1). Incubation in 25% tetrahydrofuran did not affect the overall shape of the lentikats.
However, minor disruption of the structure in the center
of lenses was observed. The pore area and diameter in
tetrahydrofuran (62.2 ± 37.3 and 10.6 ± 2.6 μm, respectively) were smaller of lenses in phosphate buffer (Fig. 5,
Table 1). The catalytic properties of the free dehalogenase
and dehalogenase lentikats (Fig. 5) differed in tetrahydrofuran. Free dehalogenase showed good activity in 5 and
10% tetrahydrofuran, whereas 20% tetrahydrofuran had a
deleterious effect on the catalytic activity; the free dehalogenase was not active under the latter concentration,
whereas the dehalogenase lentikats retained a catalytic
activity of 21%.
Tetrahydrofuran is often substituted by dioxane in
some processes because of the latter’s lower toxicity.
A negligible effect on the structure of lenses was observed
in the case of 25% dioxane (pore area and diameter of
71.4 ± 56.0 and 12.8 ± 3.0 μm, respectively, Fig. 5, Table 1),
whereas 50% dioxane caused a reduction in the elasticity
and higher incurvation of the lenses. The observed pores
were significantly smaller in the presence of 50% dioxane
(pore area and diameter of 21.6 ± 12.1 and 7.0 ± 1.4 μm,
respectively) compared to lenses incubated in phosphate
buffer (Fig. 5, Table 1). The catalytic activities of both free
dehalogenase and dehalogenase lentikats in 5 and 10%
dioxane were comparable to the activity in glycine buffer
(Fig. 5). Activities of approximately 90% were observed in
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Figure 5. Effect of organic cosolvents on
(A–D) the structure and (E–F) the activity of lentikats. Lentikats were incubated
in (A) 50 mM phosphate buffer, pH 7.5,
(B) 25% tetrahydrofuran, (C) 25% dioxane, and (D) 50% dioxane for 1 week.
The images were acquired in the center
of a lentikat using confocal microscopy
at a magnification of 400×. The pore size
distribution was determined using image
analysis of 200 pores in the center (white
column) and at the edge (black column)
of lentikats. Activity of free dehalogenase
and dehalogenase lentikats towards
bromocyclohexane was measured in
the presence of (E) tetrahydrofuran or
(F) dioxane at 25°C. The error bars show
the standard deviation of three replicate
measurements.
20% dioxane for both free dehalogenase and dehalogenase lentikats. The dehalogenase lentikats performed better in 30, 40, and 50% dioxane than the free enzyme. This
was particularly evident in the case of 50% dioxane, in
which the free enzyme was completely inactive, whereas
the dehalogenase lentikats exhibited an activity of 20%.
These results are consistent with data measured by Wilson et al. [32], who observed that penicillin G acylase
CLEAs entrapped in lentikats showed a higher activity
than the free enzyme in 75% dioxane. The authors suggested that the hydrophilic matrix surrounding the
CLEAs protected the enzyme from inactivation in organic media. This is supported by our microscopic analysis.
The narrow pores of the matrix could help to protect the
biocatalyst by hindering access of the cosolvent molecules to the CLEAs, thus lowering the deleterious effect of
the cosolvent on aggregates in the interior of lenses. This
hypothesis is also supported by obvious correlation
between activity of free enzyme and CLEAs incubated in
25% tetrahydrofuran and 50% dioxane and pore size of
polymeric matrix incubated in both cosolvents compared
to incubation in buffer.
8
4 Concluding remarks
Bright-field and electron microscopy were used to provide
an insight into the distributional and structural changes
induced during optimization of a catalyst’s immobilization. Confocal and electron microscopy enabled the structure of a biocatalyst entrapped in a porous matrix and
quality of immobilization to be analyzed. Microscopic
evaluation of the internal structure of the matrix exposed
to different organic cosolvents suggested an explanation
for the different catalytic behavior of the free and immobilized biocatalyst. The results demonstrated that microscopic techniques are useful tools obtaining information
about an immobilized biocatalyst, which can help to optimize the immobilization procedure.
The authors are grateful to Dr. Milan Balaz and Mgr.
Martin Rozmos (Masaryk University, Czech Republic) for
their imaging and documentation of CLEAs using brightfield optical microscopy. The authors also thank Dr. Radek
Stloukal (LentiKat’s a.s., Czech Republic) for introducing
us to lentikat preparation and providing the material
for immobilization. This research was supported by
funds from the Grant Agency of the Czech Republic
Biotechnology
Journal
(P207/12/0775), the Czech Ministry of Education (LO1214),
the European Regional Development Fund (CZ.1.05/
1.1.00/02.0123 and CZ.1.07/2.3.00/20.0183) and the European Union Framework Programme (REGPOT 316345).
Jiri Damborsky and Zbynek Prokop are founders of Enantis Ltd., a biotechnology spin-off company from Masaryk
University. The other authors declare no financial or commercial conflict of interest.
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9

Microscopic monitoring provides information on structure

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