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Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Surface and enzyme effects
Accepted Manuscript Title: Immobilization of Enzymes using Non-ionic Colloidal Liquid Aphrons (CLAs): Surface and Enzyme Effects. Author: Keeran Ward Jingshu Xi David C. Stuckey PII: DOI: Reference:
S0927-7765(15)30202-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.09.033 COLSUB 7373
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-5-2015 12-8-2015 18-9-2015
Please cite this article as: Keeran Ward, Jingshu Xi, David C.Stuckey, Immobilization of Enzymes using Non-ionic Colloidal Liquid Aphrons (CLAs): Surface and Enzyme Effects., Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.09.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Immobilization of Enzymes using Non-ionic Colloidal Liquid Aphrons (CLAs): Surface and Enzyme Effects. Keeran Ward, Jingshu Xi, David C Stuckey* Department of Chemical Engineering, Imperial College London, SW7 2AZ London, UK.
7
Telephone: +44 20 7594 5591; Fax: +44 20 7594 5629. Email:[email protected],
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[email protected]*
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Graphical abstract
Highlights We evaluate the immobilization capacity of Non-ionic Colloidal Liquid Aphrons. Surface adsorption strongly governed immobilization. Hydrophobic, uncharged enzymes gave a greater capacity. Cooperativity at higher concentrations was also observed.
1 2 3 4
Abstract The use of non-ionic Colloidal Liquid Aphrons (CLAs) as a support for enzyme
5
immobilization was investigated. Formulation required the mixing of an aqueous- surfactant
6
solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet
7
surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic
8
surfactants have showed increased retention, however, very little have been understood about
9
the forces governing immobilization. This study seeks to determine the effects of enzyme
10
properties on CLA immobilization by examining a non-ionic/non-polar solvent system
11
comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes
12
lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic
13
interactions strongly governed immobilization. Confocal Scanning Laser Microscopy
14
(CSLM) revealed that immobilization was predominantly achieved by surface adsorption
15
attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme
16
surface affinity was found to increase when added directly to the formulation (pre-
17
manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition),
18
with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively
19
unaffected. The effect of zeta potential on immobilization showed that enzymes adsorbed
20
better closer to their pI, indicating that charge minimization was necessary for
21
immobilization. Finally, the effect of increasing enzyme concentration in the aqueous phase
22
resulted in an increase in adsorption for all enzymes due to cooperativity between protein
23
molecules, with saturation occurring faster at higher adsorption rates.
24 25 26
Keywords: Enzyme; Surfactant; Colloidal Liquid Aphron; Immobilization; Adsorption.
1
Introduction
2 3
Enzymes are naturally occurring biocatalysts responsible for many naturally occurring
4
processes in everyday life. Due to their remarkable features, some of which include
5
specificity, high catalytic ability and stereoselectivity, it is not surprising that these
6
biocatalysts are responsible for a number of industrial reactions. With an increase in genetic
7
engineering, enzymes will soon replace conventional catalysts reducing the capital needed for
8
industrial plants. However, the industrial conditions required such as, elevated pH and
9
temperature, can affect enzyme functionality. Hence the need to preserve the catalytic
10
properties of enzymes becomes increasingly important [1] Immobilization can not only
11
secure catalytic ability, but also increase stability, robustness and in some cases can result in
12
superactivity. The main classifications of immobilization are adsorption, entrapment and
13
cross-linking [2].
14
Polyaphron systems can be effective supports for immobilization, providing a layer of bound
15
water needed for enzyme preservation. Polyaphrons are concentrated oil-in-water biliquid
16
foams stabilized by aqueous and organic surfactants, which allow a greater internal organic
17
phase to continuous aqueous phase ratio than that permissible by hexagonal close packing
18
[3]. When polyaphrons are dispersed in water they generate Colloidal Liquid Aphrons
19
(CLAs), where the 5-20μm cores are suitable for enzyme immobilization[3].Due to their
20
relative size, CLAs present large surface areas for mass transfer as well as adsorption. CLA
21
immobilization has been postulated to be governed by adsorption of the protein to the oil core
22
of the droplet [4], and exposure of proteins to surfaces and interfaces almost always leads to
23
adsorption [5]. The process of adsorption comprises various stages whereby the protein is
24
transported from the bulk phase to the sub-surface region, and due to the extent of the
25
interactions at the surface will optimise its orientation leading to structural changes. As the
1
surface coverage increases, the adsorbing molecule changes its orientation to occupy the
2
remaining spaces. Studies on many different protein systems have shown the extent of both
3
electrostatic and hydrophobic interactions facilitating adsorption [6-11].
4
Enzyme immobilization for use in bioreactors using CLAs has been researched mainly using
5
anionic surfactants, with results showing the effects of concentration and pH on
6
immobilization [4, 12-14]. However, very little work has been carried out looking at non-
7
ionic CLAs despite non-ionic surfactants being shown to be relatively gentle in nature, and
8
preserving protein conformation.
9
Hence, the aim of this investigation was to determine the forces governing non-ionic CLA
10
immobilization, and the parameters influencing successful enzyme retention. The enzymes
11
chosen for this study were both hydrophilic and hydrophobic in order to better understand the
12
interactions that promote adsorption. The effects of varying system parameters such as pH,
13
enzyme addition, enzyme concentration and surface charge were examined in an effort to
14
quantify the conditions necessary for optimum immobilization.
15 16 17 18 19 20
Materials and Methods
21
Mucopeptide N-acetylmuramylhydrolase, 90%, Sigma), Lipase (EC 3.1.1.3, Sigma) from
22
Aspergillus Niger, Aprotinin from bovine lung (Sigma) and α-Chymotrypsin from bovine
23
pancreas (3.4.21.1, Sigma). Mineral oil (Sigma), Tween 20 (Polyoxyethylene sorbitan
24
monolaurate, 99%, Sigma) and Tween 80 (Polyoxyethylene sorbitan monooleate, 99%,
25
Sigma) were used for aphron formulation. Potassium phosphate monobasic (KH2PO4, 99%
26
Sigma) and Tris-HCl (Trizma Hydrochloride, 99%, Sigma) were used for buffering. The
27
high-performance liquid chromatography grade organic solvent, acetonitrile (99.9%, VWR),
Materials Enzyme experiments were performed using lysozyme from chicken egg white (EC 3.2.1.17,
1
and reagent grade trifluoroacetic acid (99.5%, Sigma) were used for assaying lipase
2
concentration. Fluorescein isothiocyanate isomer I (FITC, Sigma) was used for protein
3
labelling, while the 660nm protein assay (ThermoScienfic) was used for a colorimetric
4
determination of aprotinin, lysozyme and α-chymotrypsin concentration. Disposable
5
desalting columns (GE Healthcare Life Sciences) were used for conjugate separation. The
6
water used throughout the experiment was distilled and deionised.
7 8 9 10
Instruments A UV-VIS scanning spectrophotometer (UV-1800 Shimadzu, UK) and High Performance
11
Liquid Chromatography (HPLC) were used for analysing enzyme samples, using a silica-
12
based C4 HPLC column (Phenomenex UK). An overhead stirrer (Heidolph RZR 2020) was
13
used for polyaphron preparation, while a Biofuge Stratos centrifuge (Heraeus Instruments)
14
was used for CLA separation. Disposable syringes (B.Braun Melsungen AG) and 0.22 μm
15
syringe filters (Millipore Co.) were purchased for the removal of particulates before sample
16
analysis. Confocal Scanning Laser Microscopy (CSLM) was performed using the LSM-700
17
(Carl Zeiss, Germany). Zeta potential measurements were taken using a ZetaPALS analyser
18
(Brookhaven Instruments), while particle size analysis was measured using the Mastersizer
19
2000 (Malvern instruments).
20 21
Protein Preparation
22
Aprotinin protein samples were prepared in 20mM potassium phosphate (monobasic) buffer,
23
while lipase was prepared using 50mM Tris HCL buffer. α-chymotrypsin samples were made
24
up via stock solutions using 1mM HCL solution consisting of 2mM CaCl2, and further
25
diluted to 0.08mM.
26
1
Polyaphron Formulation.
2
Polyaphron phases were made by the dropwise addition of the organic solvent/surfactant
3
solution (1% w/v Tween 80) from a burette into a stirred foaming aqueous phase (1% w/v
4
Tween 20 in enzyme solution) using overhead stirring until the required phase volume ratio
5
(PVR= Vorg.Vaq-1) was reached. The solvent was added at a flowrate of 0.3mL.min-1 at a
6
stirrer speed of 700 rpm; after addition of the total volume of oil, the formulation was sheared
7
using the same apparatus at 1000 rpm in order to achieve the desired particle size (~10μm).
8
The resulting formulation was very viscous with a creamy white appearance, and showed no
9
phase separation over a period of several weeks. Polyaphron formulations were made up to
10
PVR 8 and diluted to PVR 4. PVR 8 was the best choice taking into consideration viscosity
11
limitations as well as uniform particle sizes. However, PVR 8 formulations became difficult
12
for volume measurements; hence the formulation was consequently diluted for easier
13
estimations while the particle size and particle size distribution was unaffected.
14 15
CLA manufacture, Sample Preparation and Protein Assay
16
Polyaphron samples were dispersed into a bulk continuous buffer phase at a ratio of the
17
dispersed phase to the bulk of approximately 40% oil: 60%. Samples were vortex mixed for
18
1-2 minutes for homogeneity and allowed to settle for 1 hour; upon settling two distinct
19
layers were observed and the samples were centrifuged at 8500 g for 25 minutes for further
20
separation without CLA deterioration. A 1-2mL sample of the supernatant was pipetted and
21
filtered using 0.22 μm syringe filters to ensure a CLA free sample for protein assaying.
22
Aprotinin and α-chymotrypsin samples were assayed for concentration using the colorimetric
23
660nm protein assay against blank CLA dispersions. Lipase samples (100μL) were injected
24
into a Phenomenex C4 HPLC column equilibrated with 0.1% Trifluoroacetic acid (TFA) in
25
water (mobile phase A). The column was eluted using 0.1% TFA in acetonitrile (mobile
1
phase B) using a gradient from 2-30% B in 15 min, and then a ramp from 30-90% for another
2
15mins at a flowrate of 1.5 mL/min. Retention time (tR) for lipase was around 2.6 minutes.
3
Chromatograms were analysed using Origin Pro 9 software.
4
Determination of Immobilized enzyme concentration.
5
After quantification of the unbound protein in the supernatant through separation of the
6
polyaphron phase from the bulk continuous phase, a mass balance was carried out to estimate
7
the amount of protein adsorbed, as follows:
8 9
(1)
10
where, Ma is the mass immobilized [mg], Co is the enzyme loading (concentration) examined
11
[mg.mL-1], Vo is the volume of enzyme solution used [mL], Ce is the enzyme concentration in
12
the supernatant [mg.ml-1], and Vs is the volume of the supernatant [mL].
13
The amount immobilized was calculated as the mass immobilized per CLA surface as
14
described by Sebba (1987) [3]:
15
(2)
16
where, R is the radius of the CLA [m], and V is the volume of oil used for manufacture [m3].
17
Enzyme addition experiments at varying pH and Protein concentrations
18
In an effort to examine how enzyme addition affects immobilization, the enzyme was added
19
in two ways: 1) Directly to the aqueous phase of the polyaphron formulation- referred to as
20
pre-manufacture addition, and, 2) Directly to the bulk continuous phase during polyaphron
21
dispersion - referred to as post-manufacture addition. For pre-manufacture addition
22
polyaphrons were formulated as described previously, while for post-manufacture addition
23
the polyaphrons formulated were made up using only buffered aqueous phases with no
24
enzyme present. Immobilization was quantified based on the mass of enzyme adsorbed onto
1
the CLA surface. The experimental range for enzyme loading was 0.1-0.5 mg enzyme.mL
2
polyaphron-1 (pre) and 1-5mg enzyme.mL polyaphron-1 (post), while pH was kept within the
3
range of 4-9.
4 5
Error Analysis
6
Experimental errors for all results were measured by calculating the Coefficient of Variance: COV =
7 8
where σ is the standard deviation and
9
using 4 replicates giving a COV <10%.
(1)
is the mean value. Samples (n=4) were reproduced
10 11
Particle size analysis
12
The Malvern Particle Size analyser (Mastersizer 2000) was used to quantify CLA size.
13
Polyaphron samples were diluted by adding 0.1mL of the formulation directly into the
14
dispersion unit (120mL volume) filled with deionised water, stirred at 2400 rpm. For each
15
sample 6 readings were taken with the average particle size quoted D [4,3], giving a COV of
16
± 6%.
17 18
Confocal Scanning Laser Microscopy (CSLM)
19
To further understand the adsorption phenomena associated with CLAs, characterisation of
20
enzyme immobilization was facilitated by CLSM. Lysozyme was chosen as the model
21
protein since it can be easily conjugated with the fluorophore, Fluorescein isothiocyanate
22
isomer I (FITC). The use of the protease, α-chymotrypsin, was considered to assess whether
23
immobilized lysozyme affinity was stronger for the outer surface aqueous surfactant opposed
24
to the inner solvent core.
25
The LSM-700 Confocal Scanning Laser Microscope was used for imaging. An argon laser
1
was used to excite conjugates at a wavelength of 488nm using an oil-immersion objective
2
lens of 63X. The gain voltage on the photo multiplier detector was held between 965 and
3
1200V. The scan speed was held constant for all images giving a pixel dwell time of
4
12.61µsec. Scans for protease experiments were obtained under the same microscope
5
conditions and laser intensity, and images were analysed using both the Zen 2010 and 2009
6
software.
7
Fluorescein isothiocyanate isomer I (FITC) was added directly to lysozyme solutions in a
8
molar ratio of 1:10 (Enzyme: FITC) at pH 6.2 and 22 °C. The solution was mixed for 2-3
9
minutes and stored at 4°C for 24 hours. Separation of conjugate protein from FITC was
10
achieved using disposable desalting columns using the spin protocol method supplied by GE
11
Healthcare.
12
For protease experiments, 0.4mg Lysozyme. mL polyaphron-1 formulations were dispersed in
13
20mM phosphate buffer (blank) and 0.5mg.mL-1 α-chymotrypsin solutions in the ratio 60%
14
water/40% oil for 1 hour, at pH 6.2 and 22°C. Polyaphron samples were recovered after
15
centrifugation at 8500 g for 10 minutes.
16 17
Zeta Potential Measurements
18 19
Zeta potential was measured using the Brookhaven ZetaPALS analyser. Polyaphron samples
20
were made by post-enzyme addition made up using buffered blanks and an enzyme loading
21
of 4mg.mL-1 polyaphron. Polyaphron samples were diluted to 0.01mL polyaphron/mL buffer
22
solution of which 2 mL of the dispersion were analysed for zeta potential using the
23
Smoluchowski equation. For each sample 8 zeta potential readings were taken yielding a
24
COV <10%, with the mean value used as the quoted zeta potential.
25
1
Results and Discussion
2 3
Confocal Imaging
4
Previously it was found that the structure and packing of the polyaphron system changes
5
depending on the PVR, from polyhedral for systems with a PVR of 10, to ovoid for PVR
6
4[15]. To fully understand how proteins adsorb to polyaphrons, fluorescent images generated
7
from fluorescein labelled lysozyme, lipase, α-chymotrypsin and aprotinin were captured
8
using CSLM. The images (Figure 1) show a very densely packed system of spherical/ovoid
9
droplets, supporting their observations for systems made up to PVR 4. It follows that a
10
spherical arrangement would be expected for polyaphron dispersions since this organisation
11
is most thermodynamically favourable.
12
The fluorescent intensity of the images was maintained for lysozyme, showing an acceptable
13
affinity of the enzyme for the fluorophore. The images clearly show a strong affinity for the
14
polyaphron, promoting immobilization, however, the ability to determine specifically
15
whether the enzyme interacts with the oil core (encapsulated) or the ‘’soapy shell’’ (surface
16
adsorption) has still yet to be determined. Evidence of clustering can be observed between
17
adjacent aphrons, with the likelihood of clustering increasing for droplet populations with
18
lower particle size. Protein clustering, evident by the bright fluorescent patches shown in
19
Figure 1, was the result of short range hydrophobic interactions between enzyme molecules
20
upon binding to the surface.
21
In an effort to quantify the degree of enzyme affinity occurring during polyaphron
22
manufacture, lysozyme samples were dispersed into buffer and 0.5mg.mL-1 α-chymotrypsin
23
solutions. Images were taken using the same microscope conditions and laser intensity. The
24
loss of intensity comparing Figures 1 and 2A shows that upon dispersion some of the surface
25
bound lysozyme was washed off, leaving behind more defined protein clusters as well as
1
thinner protein layers. These observations are fairly consistent with those previously reported
2
[4], explaining the increase in enzyme retention after subsequent staged dispersions to be
3
most likely the effect of washing off loosely bound protein from the surface, leaving behind a
4
compact monolayer. Comparing Figures 1B and C, it can be observed that there is a direct
5
loss of florescence. As α-chymotrypsin interacts with lysozyme, the structure of the protein
6
becomes compromised as cleavage persists along the hydrophobic residues of the lysozyme
7
secondary structure, resulting in a decrease in florescence. At a concentration of 0.5mg.mL-1
8
α-chymotrypsin has very little affinity for CLAs, and hence the results suggest that protease-
9
lysozyme interactions would occur closest to the bulk phase indicating that lysozyme
10
interacted strongly with the surface. Also, due to the nature of lysozyme being highly
11
hydrophilic the enzyme would interact more strongly with the ‘soapy shell’ of the aphron
12
rather than the hydrophobic solvent core. Hence, these results aid in clarifying that
13
immobilization was strongly attributed to surface adsorption rather than encapsulation.
14
Effect of enzyme addition and pH
15
The effects of concentration and pH on adsorption are shown in Figures 2 and 3. For lipase,
16
when added directly to the polyaphron at pH 4.8 it gave particle sizes >20 µm, but upon
17
shearing the polyaphron became unstable and phase inverted. Studies on the stability of lipase
18
from Aspergillus Niger have been reported [16] within the pH range of 5-7.5, and hence can
19
explain the instability of lipase at pH 4.8. However, for post-manufacture addition
20
experiments lipase immobilization was found to be similar to those carried out at pH 7.5 and
21
8.5.
22
Comparing the curves of Figure 2 and 3, it can be observed that pre-manufacture addition
23
increases enzyme retention compared to post-manufacture addition; this was evident when
24
comparing α-chymotrypsin in both figures. Comparing the polyaphron loading, post-
25
manufacture addition results in a 10 times higher enzyme loading [mg.mL-1] compared to
1
pre-manufacture addition. Although the concentration was higher, the mass adsorbed was
2
much lower than that of pre-enzyme addition for both α-chymotrypsin and aprotinin. Also,
3
experiments carried out at similar polyaphron loadings as that of pre-manufacture addition
4
resulted in little to no immobilization. Similar results were observed by Lamb and Stuckey
5
(1999) [13] for the immobilization of β-galactosidase through post-manufacture addition at
6
similar enzyme loadings using an ionic formulation. The greater affinity for immobilization
7
when the enzyme was added directly to the polyaphron can be attributed to the close
8
proximity to the surface during formulation, allowing for enhanced hydrophobic interactions.
9
However, for lipase its extent of adsorption was not affected by addition, and this can be
10
directly attributed to its hydrophobic nature.
11
As pH increased, aprotinin was most affected compared to α-chymotrypsin and lipase. These
12
effects can be attributed to charge interactions between protein molecules within the aqueous
13
and bulk phases; α-chymotrypsin being fairly hydrophobic (Table 1) and much larger than
14
aprotinin, produces a much lower surface charge. As a result the extent of charge interactions
15
is more relaxed compared to aprotinin, leading to greater adsorption. Studies investigating
16
adsorption of a number of proteins to charged polystyrene showed that electrostatic
17
interactions played a major role in adsorption phenomena, depending mainly on protein
18
charge, pI and pH conditions [6].
19
For lipase, however, it was found that pH did not significantly affect its immobilization.
20
These results were consistent with studies investigating the effect of immobilization of
21
Candida Antarctica Lipase A on polystyrene. The results showed that immobilization was
22
generated by the hydrophobic interactions between the hydrophobic patches on the surface of
23
lipase, and the hydrophobic nature of polystyrene [7].
24 25
Effect of surface charge
1
In an effort to examine the effect of surface charge on CLA immobilization, zeta potential
2
measurements were taken for both enzyme loaded and blank CLA dispersions to account for
3
charge differences during immobilization. The results in Table 2 show largely negative zeta
4
potentials before and after immobilization, with an increasing trend observed for enzyme
5
loaded dispersions as the pH increases. The blank samples dispersed in phosphate buffered
6
systems showed limited variation in zeta potential compared to those dispersed in Tris HCl;
7
the differences between the two show the attractive nature of counterions present in solution
8
for the CLA surface. Essentially the surface of the CLA would be comprised of non-ionic
9
surfactants which create a fairly neutral hydrophobic surface, however, the presence of
10
counterions such as
ions can preferentially adsorb to the surface decreasing the zeta
11
potential greatly [17, 18]. The presence of
12
Tris HCl can lead to charge neutralisation at the surface yielding a lower zeta potential.
13
Furthermore, it has been reported that non-ionic surfactants, in particular those with the
14
sorbitan group (Tween surfactants) can possess a stable negative charge within the pH range
15
of 5-9 at concentrations above their critical micelle concentration [19]. This can further
16
explain the negative zeta potentials reported for the blank CLAs.
17
Upon immobilization, as the enzyme adsorbs to the CLA surface the overall surface charge
18
changes based on the charge density of the enzyme. To account for the affinity of the enzyme
19
for the CLA surface, the zeta potential difference between the blank and immobilised enzyme
20
formulations were calculated as shown in the bar chart in Figure 4. For most enzymes the
21
change in zeta potential was largest when the pH is furthest away from their respective pIs
22
(pH 4.8), since they are largely positively charged (pH
23
overall surface charge of the CLA from negatively to positively charged. Comparing the
24
hydrophobicity data in Table 1 and the zeta potential data in Table 2, the charge densities of
25
the enzymes can be listed in decreasing order; aprotinin > α-chymotrypsin > lipase.
ions as well as amine groups associated with
1
Furthermore, comparing the amount immobilised in Figure 2B with the zeta potential
2
differences in Figure 4, it can be clearly shown that charge density plays some role in the
3
ability of the protein to adsorb to the CLA surface. Although aprotinin yields the largest
4
charge density compared to α-chymotrypsin, its change in zeta potential was much less at pH
5
4.8. Comparing the adsorption results for both proteins at pH 4.8, the amount of α-
6
chymotrypsin adsorbed was almost 3 times that of aprotinin, and hence a higher zeta potential
7
difference was observed for α-chymotrypsin. As pH increases, charge density and repulsion
8
between protein molecules decreases allowing for greater adsorption and a general lowering
9
of the zeta potential difference due to hydrophobic interactions. However, the decrease in
10
charge density was more pronounced with α-chymotrypsin as the pH increases closely
11
towards its pI, and hence its zeta potential difference was the lowest. The effect of lipase on
12
zeta potential was fairly minimal, showing that hydrophobic interactions were responsible for
13
immobilization. Hence, it can be shown that the higher the charge density the greater the
14
repulsive forces which can lead to lower adsorption [6, 10].
15 16 17 18
Effect of concentration The results from Figures 2 and 3 generally show an increase in adsorption as concentration
19
increases. This was expected due to increased binding resulting from the availability of
20
protein molecules as concentration increases. Past research investigating enzyme
21
concentration and CLA immobilization showed that a spatial arrangement of protein
22
molecules existed on the surface at low concentrations, and were bound at specific locations
23
on the surface [4]. However, at higher concentrations, due to increased protein adsorption,
24
this arrangement shifted to a close-packed assembly of protein molecules exhibiting stronger
25
protein-protein interactions, and hence increasing immobilization. As the concentration
26
increased, the protein adsorbed formed layers on the surface resulting in multilayers.
27
However, the proximity to the surface decreases as the layers grew, hence the forces
1
governing adsorption decreases gradually resulting in saturation. For concentrated protein
2
systems, the surface area occupied by the protein upon adsorbing increases with the residence
3
time at the surface; a process called spreading[20]. For a high initial adsorption rate the time
4
allowed for spreading is lower, allowing for more molecules to be accommodated on the
5
same surface area, resulting in an increase in adsorption saturation with concentration.
6
Although the overall enzyme concentration during post-manufacture addition is much higher
7
than that of pre-manufacture addition, the saturation point was reached faster during pre-
8
manufacture addition indicating a faster spreading rate. This can be directly attributed to the
9
binding affinity between neighbouring protein molecules and the competition for available
10
surface, resulting in increased saturation. As this occurs the strength of the protein- protein
11
interactions at the surface is lower than that of the bulk allowing for the displacement of
12
protein from the surface, as shown in Figure 2.
13
The results also show that nonlinear adsorption occurs more when the enzyme was added
14
directly to the polyaphron, rather than when it was added to the bulk continuous phase. This
15
nonlinearity arises from cooperativity due to protein-protein interactions occurring as the
16
initial adsorption rate increases. These interactions are increased by the large affinity for
17
adsorption arising from direct enzyme addition. Cooperative adsorption is the result of
18
clustering arising from the self-organisation of the adsorbed monomers, and the interactions
19
between those molecules and new incoming molecules from the bulk phase. One study
20
exploring cooperative adsorption of proteins onto solid surfaces found that interactions
21
between adsorbed proteins results in minute changes in density, evolving into persistent
22
density transitions after continued adsorption; this leads to the formation of protein patches
23
next to loosely covered surface regions [21]. Another study examined a cluster theory behind
24
protein adsorption, investigating three pathways; direct adsorption onto the surface, accretion
25
of the adsorbed monomer into the cluster, and piggyback deposition allowing for
1
incorporation of the adsorbed monomer into the cluster [22]. They found that in the absence
2
of direct adsorption the rate of adsorption will always be less than ideal due to the overall
3
decrease in available surface area. When adsorption is coupled with clustering, several
4
regimes are observed based on the rate of direct adsorption and the growth of clustering
5
resulting in cooperativity. The onset of clustering has been proven to be the direct result of
6
electrostatic interactions as well as interactions due to an increased binding affinity resulting
7
from conformational changes. For charged surfaces initial adsorption is driven by coulombic
8
interactions with the surface [23], with clustering being formed by electrostatic interactions
9
with the resulting electric field generated by pre-adsorbed proteins. However, for
10
hydrophobic surfaces initial adsorption is thought to be driven by hydrophobic interactions
11
with clustering being supported by lateral interactions between adsorbed proteins, as well as
12
increased hydrophobic affinity due to possible conformational change.
13 14
Conclusion
15
An investigation into the potential use of CLAs for enzyme immobilization was conducted
16
using both hydrophilic (aprotinin) and hydrophobic (lipase and α-chymotrypsin) enzymes.
17
Through the use of CSLM, the immobilization was predominantly achieved through surface
18
adsorption. Insights into protease degradation using α-chymotrypsin showed that lysozyme
19
affinity for the outer aqueous layer of the aphron was much greater than the inner solvent
20
core. Also, by analyzing the zeta potential of the loaded CLAs, it was found that
21
immobilization greatly decreased the surface charge of the CLA owing to enzyme adsorption
22
onto the CLA surface. Furthermore, adsorption of the charged proteins aprotinin and α-
23
chymotrypsin was found to increase when the pH was closest to their respective pIs,
24
indicating that repulsive forces between free protein molecules hindered immobilization.
25
Examination of adsorption profiles showed that hydrophobic interactions were the main force
1
contributing to adsorption. This was verified as hydrophobic enzymes, lipase and α-
2
chymotrypsin, gave the best performance. The effect of increased enzyme concentration
3
showed a direct increase in immobilization, with saturation being observed faster when the
4
enzyme was added directly to the formulation due to higher initial adsorption rates resulting
5
from increased surface affinity, higher induced protein-protein interactions and cooperativity.
6 7 8
Acknowledgments
9
The authors are grateful for the partial funding received from MC2 Biotek, Derek Wheeler,
10
Stephen Lenon and Fraser Steele from Drug Delivery Solutions for their insight, and to
11
Christine Seifried for obtaining the confocal microscope images. The microscope is located
12
in the Qatar Carbonates and Carbon Storage Research Centre and was purchased using
13
funding from Qatar Petroleum, Shell and Qatar Science and Technology Park.
14
1 2 3
List of References.
4 5
[1] S.B. Lamb, Enzyme Immobilization on Colloidal Liquid Aphrons (CLAs) and the
6
Development of a Continuous Membrane Bioreactor, Imperial College London, 1999.
7
[2] G. Bickerstaff, Immobilization of Enzymes and Cells, Humana Press Inc1997.
8
[3] F. Sebba, Foams and Biliquid foams- Aphrons, John Wiley & Sons Ltd.1987.
9
[4] G.J. Lye, M. Rosjidi, O.P. Pavlou, D.C. Stuckey, Immobilization of Candida cylindracea
10
lipase on colloidal liquid aphrons (CLAs) and development of a continuous CLA-membrane
11
reactor, Biotechnol.Bioeng., 51 (1996) 69-78.
12
[5] W. Norde, C.E. Giacomelli, Conformational changes in proteins at interfaces: from
13
solution to the interface, and back, Macromol Symp, 145 (1999) 125-136.
14
[6] M. Kleijn, W. Norde, The adsorption of proteins from aqueous solution on solid surfaces,
15
Heterogen Chem Rev, 2 (1995) 157-172.
16
[7] N. Miletic, V. Abetz, K. Ebert, K. Loos, Immobilization of Candida antarctica lipase B on
17
Polystyrene Nanoparticles, Macromol Rapid Comm, 31 (2010) 71-74.
18
[8] W. Norde, C.E. Giacomelli, BSA structural changes during homomolecular exchange
19
between the adsorbed and the dissolved states, J Biotechnol, 79 (2000) 259-268.
20
[9] W. Norde, F. Macritchie, G. Nowicka, J. Lyklema, Protein Adsorption at Solid Liquid
21
Interfaces - Reversibility and Conformation Aspects, J.Colloid Interface Sci., 112 (1986) 447.
22
[10] M. Wahlgren, T. Arnebrant, Protein Adsorption to Solid-Surfaces, Trends Biotechnol, 9
23
(1991) 201-208.
24
[11] D.T.H. Wassell, G. Embery, Adsorption of Bovine Serum-Albumin to Titanium Powder,
25
J Dent Res, 74 (1995) 836-836.
1
[12] S.B. Lamb, D.C. Stuckey, Enzyme immobilization on colloidal liquid aphrons (CLAs):
2
the influence of system parameters on activity, Enzyme Microb.Technol., 26 (2000) 574-581.
3
[13] S.B. Lamb, D.C. Stuckey, Enzyme immobilisation on colloidal liquid aphrons (CLAs):
4
the influence of protein properties, Enzyme Microb.Technol., 24 (1999) 541-548.
5
[14] G.J. Lye, Stereoselective Hydrolysis of DL-Phenylalanine methyl ester and Separation
6
of L-Phenylalanine
7
Techniques, 11 (1997) 611-616.
8
[15] G.J. Lye, D.C. Stuckey, Structure and stability of colloidal liquid aphrons, Colloids and
9
surfaces.A, Physicochemical and engineering aspects, 131 (1998) 119-136.
using
aphron
immobilised
alpha-chymotrypsin,
Biotechnology
10
[16] D. Chakravorty, S. Parameswaran, V.K. Dubey, S. Patra, Unraveling the Rationale
11
Behind Organic Solvent Stability of Lipases, Appl Biochem Biotech, 167 (2012) 439-461.
12
[17] V.B. Junyaprasert, V. Teeranachaideekul, T. Supaperm, Effect of Charged and Non-
13
ionic Membrane Additives on Physicochemical Properties and Stability of Niosomes, Aaps
14
Pharmscitech, 9 (2008) 851-859.
15
[18] H. Sis, M. Birinci, Effect of nonionic and ionic surfactants on zeta potential and
16
dispersion properties of carbon black powders, Colloid Surface A, 341 (2009) 60-67.
17
[19] M. Vasudevan, J.M. Wiencek, Mechanism of the extraction of proteins into Tween 85
18
nonionic microemulsions, Ind Eng Chem Res, 35 (1996) 1085-1089.
19
[20] M. van der Veen, M.C. Stuart, W. Norde, Spreading of proteins and its effect on
20
adsorption and desorption kinetics, Colloid Surface B, 54 (2007) 136-142.
21
[21] M. Rabe, D. Verdes, S. Seeger, Understanding Cooperative Protein Adsorption Events at
22
the Microscopic Scale: A Comparison between Experimental Data and Monte Carlo
23
Simulations, J Phys Chem B, 114 (2010) 5862-5869.
24
[22] A.P. Minton, Effects of excluded surface area and adsorbate clustering on surface
25
adsorption of proteins. II. Kinetic models, Biophys.J., 80 (2001) 1641-1648.
1
[23] A.L. Creagh, J.M. Prausnitz, H.W. Blanch, Structural and Catalytic Properties of
2
Enzymes in Reverse Micelles, Enzyme Microb.Technol., 15 (1993) 383-392.
3
[24] K. Gekko, Y. Hasegawa, Compressibility Structure Relationship of Globular-Proteins,
4
Biochemistry (N.Y.), 25 (1986) 6563-6571.
5 6
1 2
List of Figures
3 4
Figure 1: Lysozyme-FITC fluorescence images of polyaphron manufactured at PVR 4. A)
5
Before polyaphron dispersion, B) After polyaphron dispersion, C) After proteolysis.
6
Excitation wavelength 488nm; magnification x63, laser intensity 4.5 %.
7
Figure 2: Adsorption profile for pre-manufacture enzyme addition as function of increasing
8
enzyme concentration at PVR 4 and 22°C. A) pH 4.8; B) pH =7.5 (Lipase-6.2), and C)
9
pH=8.5. Error bars are reported SD, n=4.
10
Figure 3: Adsorption profile for post-manufacture enzyme addition as function of increasing
11
enzyme concentration at PVR 4 and 22°C. A) pH 4.8; B) pH =7.5 (Lipase-6.2), and C)
12
pH=8.5. Error bars are reported SD, n=4.
13
Figure 4: Zeta potential difference recorded after enzyme immobilization over the pH range
14
4-9. Analysis carried out at ambient temperature 22°C. Error bars are reported SD, n=3.
15 16
1 2
List of Tables
3 4 5 6 7 8 9 10 11 12 13 14 15 16
Table 1: Enzyme properties a
Enzyme
pI
Molecular Weight[Da]
Lipase
4.1
45000
Aprotinin
10.5
6512
910 -
α-Chymotrypsin
8.75
25000
908
Hydrophobicity [cal/residue] b
a-Total hydrophobicity [24] b- Estimated hydrophobicity [13] 17 18 19
Table 2: Average zeta potential measurements taken before and after enzyme immobilization
20
(Error: SD, n=3) Formulation
Zeta Potential (mV) pH 4.8 pH 6.2/7.5 Before Immobilization
pH 8.6
Blank (phosphate)
-19.31±0.96
-23.96±1.91
-24.95±1.24
Blank (Tris HCl)
-10.61±0.98
-17.17±0.96
-20.62±1.15
After Immobilization
21 22 23
Lipase
-6.97±0.65
-12.46±0.95
-16.17±1.01
Aprotinin
-12.36±0.84
-18.57±1.02
-23.40±1.81
α-Chymotrypsin
-9.20±0.69
-23.45±1.55
-23.63±0.98
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 3
1 2 3
4
Figure 4
S0927-7765(15)30202-2 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.09.033 COLSUB 7373
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
6-5-2015 12-8-2015 18-9-2015
Please cite this article as: Keeran Ward, Jingshu Xi, David C.Stuckey, Immobilization of Enzymes using Non-ionic Colloidal Liquid Aphrons (CLAs): Surface and Enzyme Effects., Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.09.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1 2 3 4 5 6
Immobilization of Enzymes using Non-ionic Colloidal Liquid Aphrons (CLAs): Surface and Enzyme Effects. Keeran Ward, Jingshu Xi, David C Stuckey* Department of Chemical Engineering, Imperial College London, SW7 2AZ London, UK.
7
Telephone: +44 20 7594 5591; Fax: +44 20 7594 5629. Email:[email protected],
8
[email protected]*
9 10 11 12 13 14 15 16 17 18
Graphical abstract
Highlights We evaluate the immobilization capacity of Non-ionic Colloidal Liquid Aphrons. Surface adsorption strongly governed immobilization. Hydrophobic, uncharged enzymes gave a greater capacity. Cooperativity at higher concentrations was also observed.
1 2 3 4
Abstract The use of non-ionic Colloidal Liquid Aphrons (CLAs) as a support for enzyme
5
immobilization was investigated. Formulation required the mixing of an aqueous- surfactant
6
solution with a relatively non-polar solvent-surfactant solution, forming a solvent droplet
7
surrounded by a thin stabilised aqueous film (soapy shell). Studies utilising anionic
8
surfactants have showed increased retention, however, very little have been understood about
9
the forces governing immobilization. This study seeks to determine the effects of enzyme
10
properties on CLA immobilization by examining a non-ionic/non-polar solvent system
11
comprised of two non-ionic surfactants, Tween 20 and 80, mineral oil and the enzymes
12
lipase, aprotinin and α-chymotrypsin. From these results it was deduced that hydrophobic
13
interactions strongly governed immobilization. Confocal Scanning Laser Microscopy
14
(CSLM) revealed that immobilization was predominantly achieved by surface adsorption
15
attributed to hydrophobic interactions between the enzyme and the CLA surface. Enzyme
16
surface affinity was found to increase when added directly to the formulation (pre-
17
manufacture addition), as opposed to the bulk continuous phase (post-manufacture addition),
18
with α-chymotrypsin and aprotinin being the most perturbed, while lipase was relatively
19
unaffected. The effect of zeta potential on immobilization showed that enzymes adsorbed
20
better closer to their pI, indicating that charge minimization was necessary for
21
immobilization. Finally, the effect of increasing enzyme concentration in the aqueous phase
22
resulted in an increase in adsorption for all enzymes due to cooperativity between protein
23
molecules, with saturation occurring faster at higher adsorption rates.
24 25 26
Keywords: Enzyme; Surfactant; Colloidal Liquid Aphron; Immobilization; Adsorption.
1
Introduction
2 3
Enzymes are naturally occurring biocatalysts responsible for many naturally occurring
4
processes in everyday life. Due to their remarkable features, some of which include
5
specificity, high catalytic ability and stereoselectivity, it is not surprising that these
6
biocatalysts are responsible for a number of industrial reactions. With an increase in genetic
7
engineering, enzymes will soon replace conventional catalysts reducing the capital needed for
8
industrial plants. However, the industrial conditions required such as, elevated pH and
9
temperature, can affect enzyme functionality. Hence the need to preserve the catalytic
10
properties of enzymes becomes increasingly important [1] Immobilization can not only
11
secure catalytic ability, but also increase stability, robustness and in some cases can result in
12
superactivity. The main classifications of immobilization are adsorption, entrapment and
13
cross-linking [2].
14
Polyaphron systems can be effective supports for immobilization, providing a layer of bound
15
water needed for enzyme preservation. Polyaphrons are concentrated oil-in-water biliquid
16
foams stabilized by aqueous and organic surfactants, which allow a greater internal organic
17
phase to continuous aqueous phase ratio than that permissible by hexagonal close packing
18
[3]. When polyaphrons are dispersed in water they generate Colloidal Liquid Aphrons
19
(CLAs), where the 5-20μm cores are suitable for enzyme immobilization[3].Due to their
20
relative size, CLAs present large surface areas for mass transfer as well as adsorption. CLA
21
immobilization has been postulated to be governed by adsorption of the protein to the oil core
22
of the droplet [4], and exposure of proteins to surfaces and interfaces almost always leads to
23
adsorption [5]. The process of adsorption comprises various stages whereby the protein is
24
transported from the bulk phase to the sub-surface region, and due to the extent of the
25
interactions at the surface will optimise its orientation leading to structural changes. As the
1
surface coverage increases, the adsorbing molecule changes its orientation to occupy the
2
remaining spaces. Studies on many different protein systems have shown the extent of both
3
electrostatic and hydrophobic interactions facilitating adsorption [6-11].
4
Enzyme immobilization for use in bioreactors using CLAs has been researched mainly using
5
anionic surfactants, with results showing the effects of concentration and pH on
6
immobilization [4, 12-14]. However, very little work has been carried out looking at non-
7
ionic CLAs despite non-ionic surfactants being shown to be relatively gentle in nature, and
8
preserving protein conformation.
9
Hence, the aim of this investigation was to determine the forces governing non-ionic CLA
10
immobilization, and the parameters influencing successful enzyme retention. The enzymes
11
chosen for this study were both hydrophilic and hydrophobic in order to better understand the
12
interactions that promote adsorption. The effects of varying system parameters such as pH,
13
enzyme addition, enzyme concentration and surface charge were examined in an effort to
14
quantify the conditions necessary for optimum immobilization.
15 16 17 18 19 20
Materials and Methods
21
Mucopeptide N-acetylmuramylhydrolase, 90%, Sigma), Lipase (EC 3.1.1.3, Sigma) from
22
Aspergillus Niger, Aprotinin from bovine lung (Sigma) and α-Chymotrypsin from bovine
23
pancreas (3.4.21.1, Sigma). Mineral oil (Sigma), Tween 20 (Polyoxyethylene sorbitan
24
monolaurate, 99%, Sigma) and Tween 80 (Polyoxyethylene sorbitan monooleate, 99%,
25
Sigma) were used for aphron formulation. Potassium phosphate monobasic (KH2PO4, 99%
26
Sigma) and Tris-HCl (Trizma Hydrochloride, 99%, Sigma) were used for buffering. The
27
high-performance liquid chromatography grade organic solvent, acetonitrile (99.9%, VWR),
Materials Enzyme experiments were performed using lysozyme from chicken egg white (EC 3.2.1.17,
1
and reagent grade trifluoroacetic acid (99.5%, Sigma) were used for assaying lipase
2
concentration. Fluorescein isothiocyanate isomer I (FITC, Sigma) was used for protein
3
labelling, while the 660nm protein assay (ThermoScienfic) was used for a colorimetric
4
determination of aprotinin, lysozyme and α-chymotrypsin concentration. Disposable
5
desalting columns (GE Healthcare Life Sciences) were used for conjugate separation. The
6
water used throughout the experiment was distilled and deionised.
7 8 9 10
Instruments A UV-VIS scanning spectrophotometer (UV-1800 Shimadzu, UK) and High Performance
11
Liquid Chromatography (HPLC) were used for analysing enzyme samples, using a silica-
12
based C4 HPLC column (Phenomenex UK). An overhead stirrer (Heidolph RZR 2020) was
13
used for polyaphron preparation, while a Biofuge Stratos centrifuge (Heraeus Instruments)
14
was used for CLA separation. Disposable syringes (B.Braun Melsungen AG) and 0.22 μm
15
syringe filters (Millipore Co.) were purchased for the removal of particulates before sample
16
analysis. Confocal Scanning Laser Microscopy (CSLM) was performed using the LSM-700
17
(Carl Zeiss, Germany). Zeta potential measurements were taken using a ZetaPALS analyser
18
(Brookhaven Instruments), while particle size analysis was measured using the Mastersizer
19
2000 (Malvern instruments).
20 21
Protein Preparation
22
Aprotinin protein samples were prepared in 20mM potassium phosphate (monobasic) buffer,
23
while lipase was prepared using 50mM Tris HCL buffer. α-chymotrypsin samples were made
24
up via stock solutions using 1mM HCL solution consisting of 2mM CaCl2, and further
25
diluted to 0.08mM.
26
1
Polyaphron Formulation.
2
Polyaphron phases were made by the dropwise addition of the organic solvent/surfactant
3
solution (1% w/v Tween 80) from a burette into a stirred foaming aqueous phase (1% w/v
4
Tween 20 in enzyme solution) using overhead stirring until the required phase volume ratio
5
(PVR= Vorg.Vaq-1) was reached. The solvent was added at a flowrate of 0.3mL.min-1 at a
6
stirrer speed of 700 rpm; after addition of the total volume of oil, the formulation was sheared
7
using the same apparatus at 1000 rpm in order to achieve the desired particle size (~10μm).
8
The resulting formulation was very viscous with a creamy white appearance, and showed no
9
phase separation over a period of several weeks. Polyaphron formulations were made up to
10
PVR 8 and diluted to PVR 4. PVR 8 was the best choice taking into consideration viscosity
11
limitations as well as uniform particle sizes. However, PVR 8 formulations became difficult
12
for volume measurements; hence the formulation was consequently diluted for easier
13
estimations while the particle size and particle size distribution was unaffected.
14 15
CLA manufacture, Sample Preparation and Protein Assay
16
Polyaphron samples were dispersed into a bulk continuous buffer phase at a ratio of the
17
dispersed phase to the bulk of approximately 40% oil: 60%. Samples were vortex mixed for
18
1-2 minutes for homogeneity and allowed to settle for 1 hour; upon settling two distinct
19
layers were observed and the samples were centrifuged at 8500 g for 25 minutes for further
20
separation without CLA deterioration. A 1-2mL sample of the supernatant was pipetted and
21
filtered using 0.22 μm syringe filters to ensure a CLA free sample for protein assaying.
22
Aprotinin and α-chymotrypsin samples were assayed for concentration using the colorimetric
23
660nm protein assay against blank CLA dispersions. Lipase samples (100μL) were injected
24
into a Phenomenex C4 HPLC column equilibrated with 0.1% Trifluoroacetic acid (TFA) in
25
water (mobile phase A). The column was eluted using 0.1% TFA in acetonitrile (mobile
1
phase B) using a gradient from 2-30% B in 15 min, and then a ramp from 30-90% for another
2
15mins at a flowrate of 1.5 mL/min. Retention time (tR) for lipase was around 2.6 minutes.
3
Chromatograms were analysed using Origin Pro 9 software.
4
Determination of Immobilized enzyme concentration.
5
After quantification of the unbound protein in the supernatant through separation of the
6
polyaphron phase from the bulk continuous phase, a mass balance was carried out to estimate
7
the amount of protein adsorbed, as follows:
8 9
(1)
10
where, Ma is the mass immobilized [mg], Co is the enzyme loading (concentration) examined
11
[mg.mL-1], Vo is the volume of enzyme solution used [mL], Ce is the enzyme concentration in
12
the supernatant [mg.ml-1], and Vs is the volume of the supernatant [mL].
13
The amount immobilized was calculated as the mass immobilized per CLA surface as
14
described by Sebba (1987) [3]:
15
(2)
16
where, R is the radius of the CLA [m], and V is the volume of oil used for manufacture [m3].
17
Enzyme addition experiments at varying pH and Protein concentrations
18
In an effort to examine how enzyme addition affects immobilization, the enzyme was added
19
in two ways: 1) Directly to the aqueous phase of the polyaphron formulation- referred to as
20
pre-manufacture addition, and, 2) Directly to the bulk continuous phase during polyaphron
21
dispersion - referred to as post-manufacture addition. For pre-manufacture addition
22
polyaphrons were formulated as described previously, while for post-manufacture addition
23
the polyaphrons formulated were made up using only buffered aqueous phases with no
24
enzyme present. Immobilization was quantified based on the mass of enzyme adsorbed onto
1
the CLA surface. The experimental range for enzyme loading was 0.1-0.5 mg enzyme.mL
2
polyaphron-1 (pre) and 1-5mg enzyme.mL polyaphron-1 (post), while pH was kept within the
3
range of 4-9.
4 5
Error Analysis
6
Experimental errors for all results were measured by calculating the Coefficient of Variance: COV =
7 8
where σ is the standard deviation and
9
using 4 replicates giving a COV <10%.
(1)
is the mean value. Samples (n=4) were reproduced
10 11
Particle size analysis
12
The Malvern Particle Size analyser (Mastersizer 2000) was used to quantify CLA size.
13
Polyaphron samples were diluted by adding 0.1mL of the formulation directly into the
14
dispersion unit (120mL volume) filled with deionised water, stirred at 2400 rpm. For each
15
sample 6 readings were taken with the average particle size quoted D [4,3], giving a COV of
16
± 6%.
17 18
Confocal Scanning Laser Microscopy (CSLM)
19
To further understand the adsorption phenomena associated with CLAs, characterisation of
20
enzyme immobilization was facilitated by CLSM. Lysozyme was chosen as the model
21
protein since it can be easily conjugated with the fluorophore, Fluorescein isothiocyanate
22
isomer I (FITC). The use of the protease, α-chymotrypsin, was considered to assess whether
23
immobilized lysozyme affinity was stronger for the outer surface aqueous surfactant opposed
24
to the inner solvent core.
25
The LSM-700 Confocal Scanning Laser Microscope was used for imaging. An argon laser
1
was used to excite conjugates at a wavelength of 488nm using an oil-immersion objective
2
lens of 63X. The gain voltage on the photo multiplier detector was held between 965 and
3
1200V. The scan speed was held constant for all images giving a pixel dwell time of
4
12.61µsec. Scans for protease experiments were obtained under the same microscope
5
conditions and laser intensity, and images were analysed using both the Zen 2010 and 2009
6
software.
7
Fluorescein isothiocyanate isomer I (FITC) was added directly to lysozyme solutions in a
8
molar ratio of 1:10 (Enzyme: FITC) at pH 6.2 and 22 °C. The solution was mixed for 2-3
9
minutes and stored at 4°C for 24 hours. Separation of conjugate protein from FITC was
10
achieved using disposable desalting columns using the spin protocol method supplied by GE
11
Healthcare.
12
For protease experiments, 0.4mg Lysozyme. mL polyaphron-1 formulations were dispersed in
13
20mM phosphate buffer (blank) and 0.5mg.mL-1 α-chymotrypsin solutions in the ratio 60%
14
water/40% oil for 1 hour, at pH 6.2 and 22°C. Polyaphron samples were recovered after
15
centrifugation at 8500 g for 10 minutes.
16 17
Zeta Potential Measurements
18 19
Zeta potential was measured using the Brookhaven ZetaPALS analyser. Polyaphron samples
20
were made by post-enzyme addition made up using buffered blanks and an enzyme loading
21
of 4mg.mL-1 polyaphron. Polyaphron samples were diluted to 0.01mL polyaphron/mL buffer
22
solution of which 2 mL of the dispersion were analysed for zeta potential using the
23
Smoluchowski equation. For each sample 8 zeta potential readings were taken yielding a
24
COV <10%, with the mean value used as the quoted zeta potential.
25
1
Results and Discussion
2 3
Confocal Imaging
4
Previously it was found that the structure and packing of the polyaphron system changes
5
depending on the PVR, from polyhedral for systems with a PVR of 10, to ovoid for PVR
6
4[15]. To fully understand how proteins adsorb to polyaphrons, fluorescent images generated
7
from fluorescein labelled lysozyme, lipase, α-chymotrypsin and aprotinin were captured
8
using CSLM. The images (Figure 1) show a very densely packed system of spherical/ovoid
9
droplets, supporting their observations for systems made up to PVR 4. It follows that a
10
spherical arrangement would be expected for polyaphron dispersions since this organisation
11
is most thermodynamically favourable.
12
The fluorescent intensity of the images was maintained for lysozyme, showing an acceptable
13
affinity of the enzyme for the fluorophore. The images clearly show a strong affinity for the
14
polyaphron, promoting immobilization, however, the ability to determine specifically
15
whether the enzyme interacts with the oil core (encapsulated) or the ‘’soapy shell’’ (surface
16
adsorption) has still yet to be determined. Evidence of clustering can be observed between
17
adjacent aphrons, with the likelihood of clustering increasing for droplet populations with
18
lower particle size. Protein clustering, evident by the bright fluorescent patches shown in
19
Figure 1, was the result of short range hydrophobic interactions between enzyme molecules
20
upon binding to the surface.
21
In an effort to quantify the degree of enzyme affinity occurring during polyaphron
22
manufacture, lysozyme samples were dispersed into buffer and 0.5mg.mL-1 α-chymotrypsin
23
solutions. Images were taken using the same microscope conditions and laser intensity. The
24
loss of intensity comparing Figures 1 and 2A shows that upon dispersion some of the surface
25
bound lysozyme was washed off, leaving behind more defined protein clusters as well as
1
thinner protein layers. These observations are fairly consistent with those previously reported
2
[4], explaining the increase in enzyme retention after subsequent staged dispersions to be
3
most likely the effect of washing off loosely bound protein from the surface, leaving behind a
4
compact monolayer. Comparing Figures 1B and C, it can be observed that there is a direct
5
loss of florescence. As α-chymotrypsin interacts with lysozyme, the structure of the protein
6
becomes compromised as cleavage persists along the hydrophobic residues of the lysozyme
7
secondary structure, resulting in a decrease in florescence. At a concentration of 0.5mg.mL-1
8
α-chymotrypsin has very little affinity for CLAs, and hence the results suggest that protease-
9
lysozyme interactions would occur closest to the bulk phase indicating that lysozyme
10
interacted strongly with the surface. Also, due to the nature of lysozyme being highly
11
hydrophilic the enzyme would interact more strongly with the ‘soapy shell’ of the aphron
12
rather than the hydrophobic solvent core. Hence, these results aid in clarifying that
13
immobilization was strongly attributed to surface adsorption rather than encapsulation.
14
Effect of enzyme addition and pH
15
The effects of concentration and pH on adsorption are shown in Figures 2 and 3. For lipase,
16
when added directly to the polyaphron at pH 4.8 it gave particle sizes >20 µm, but upon
17
shearing the polyaphron became unstable and phase inverted. Studies on the stability of lipase
18
from Aspergillus Niger have been reported [16] within the pH range of 5-7.5, and hence can
19
explain the instability of lipase at pH 4.8. However, for post-manufacture addition
20
experiments lipase immobilization was found to be similar to those carried out at pH 7.5 and
21
8.5.
22
Comparing the curves of Figure 2 and 3, it can be observed that pre-manufacture addition
23
increases enzyme retention compared to post-manufacture addition; this was evident when
24
comparing α-chymotrypsin in both figures. Comparing the polyaphron loading, post-
25
manufacture addition results in a 10 times higher enzyme loading [mg.mL-1] compared to
1
pre-manufacture addition. Although the concentration was higher, the mass adsorbed was
2
much lower than that of pre-enzyme addition for both α-chymotrypsin and aprotinin. Also,
3
experiments carried out at similar polyaphron loadings as that of pre-manufacture addition
4
resulted in little to no immobilization. Similar results were observed by Lamb and Stuckey
5
(1999) [13] for the immobilization of β-galactosidase through post-manufacture addition at
6
similar enzyme loadings using an ionic formulation. The greater affinity for immobilization
7
when the enzyme was added directly to the polyaphron can be attributed to the close
8
proximity to the surface during formulation, allowing for enhanced hydrophobic interactions.
9
However, for lipase its extent of adsorption was not affected by addition, and this can be
10
directly attributed to its hydrophobic nature.
11
As pH increased, aprotinin was most affected compared to α-chymotrypsin and lipase. These
12
effects can be attributed to charge interactions between protein molecules within the aqueous
13
and bulk phases; α-chymotrypsin being fairly hydrophobic (Table 1) and much larger than
14
aprotinin, produces a much lower surface charge. As a result the extent of charge interactions
15
is more relaxed compared to aprotinin, leading to greater adsorption. Studies investigating
16
adsorption of a number of proteins to charged polystyrene showed that electrostatic
17
interactions played a major role in adsorption phenomena, depending mainly on protein
18
charge, pI and pH conditions [6].
19
For lipase, however, it was found that pH did not significantly affect its immobilization.
20
These results were consistent with studies investigating the effect of immobilization of
21
Candida Antarctica Lipase A on polystyrene. The results showed that immobilization was
22
generated by the hydrophobic interactions between the hydrophobic patches on the surface of
23
lipase, and the hydrophobic nature of polystyrene [7].
24 25
Effect of surface charge
1
In an effort to examine the effect of surface charge on CLA immobilization, zeta potential
2
measurements were taken for both enzyme loaded and blank CLA dispersions to account for
3
charge differences during immobilization. The results in Table 2 show largely negative zeta
4
potentials before and after immobilization, with an increasing trend observed for enzyme
5
loaded dispersions as the pH increases. The blank samples dispersed in phosphate buffered
6
systems showed limited variation in zeta potential compared to those dispersed in Tris HCl;
7
the differences between the two show the attractive nature of counterions present in solution
8
for the CLA surface. Essentially the surface of the CLA would be comprised of non-ionic
9
surfactants which create a fairly neutral hydrophobic surface, however, the presence of
10
counterions such as
ions can preferentially adsorb to the surface decreasing the zeta
11
potential greatly [17, 18]. The presence of
12
Tris HCl can lead to charge neutralisation at the surface yielding a lower zeta potential.
13
Furthermore, it has been reported that non-ionic surfactants, in particular those with the
14
sorbitan group (Tween surfactants) can possess a stable negative charge within the pH range
15
of 5-9 at concentrations above their critical micelle concentration [19]. This can further
16
explain the negative zeta potentials reported for the blank CLAs.
17
Upon immobilization, as the enzyme adsorbs to the CLA surface the overall surface charge
18
changes based on the charge density of the enzyme. To account for the affinity of the enzyme
19
for the CLA surface, the zeta potential difference between the blank and immobilised enzyme
20
formulations were calculated as shown in the bar chart in Figure 4. For most enzymes the
21
change in zeta potential was largest when the pH is furthest away from their respective pIs
22
(pH 4.8), since they are largely positively charged (pH
23
overall surface charge of the CLA from negatively to positively charged. Comparing the
24
hydrophobicity data in Table 1 and the zeta potential data in Table 2, the charge densities of
25
the enzymes can be listed in decreasing order; aprotinin > α-chymotrypsin > lipase.
ions as well as amine groups associated with
1
Furthermore, comparing the amount immobilised in Figure 2B with the zeta potential
2
differences in Figure 4, it can be clearly shown that charge density plays some role in the
3
ability of the protein to adsorb to the CLA surface. Although aprotinin yields the largest
4
charge density compared to α-chymotrypsin, its change in zeta potential was much less at pH
5
4.8. Comparing the adsorption results for both proteins at pH 4.8, the amount of α-
6
chymotrypsin adsorbed was almost 3 times that of aprotinin, and hence a higher zeta potential
7
difference was observed for α-chymotrypsin. As pH increases, charge density and repulsion
8
between protein molecules decreases allowing for greater adsorption and a general lowering
9
of the zeta potential difference due to hydrophobic interactions. However, the decrease in
10
charge density was more pronounced with α-chymotrypsin as the pH increases closely
11
towards its pI, and hence its zeta potential difference was the lowest. The effect of lipase on
12
zeta potential was fairly minimal, showing that hydrophobic interactions were responsible for
13
immobilization. Hence, it can be shown that the higher the charge density the greater the
14
repulsive forces which can lead to lower adsorption [6, 10].
15 16 17 18
Effect of concentration The results from Figures 2 and 3 generally show an increase in adsorption as concentration
19
increases. This was expected due to increased binding resulting from the availability of
20
protein molecules as concentration increases. Past research investigating enzyme
21
concentration and CLA immobilization showed that a spatial arrangement of protein
22
molecules existed on the surface at low concentrations, and were bound at specific locations
23
on the surface [4]. However, at higher concentrations, due to increased protein adsorption,
24
this arrangement shifted to a close-packed assembly of protein molecules exhibiting stronger
25
protein-protein interactions, and hence increasing immobilization. As the concentration
26
increased, the protein adsorbed formed layers on the surface resulting in multilayers.
27
However, the proximity to the surface decreases as the layers grew, hence the forces
1
governing adsorption decreases gradually resulting in saturation. For concentrated protein
2
systems, the surface area occupied by the protein upon adsorbing increases with the residence
3
time at the surface; a process called spreading[20]. For a high initial adsorption rate the time
4
allowed for spreading is lower, allowing for more molecules to be accommodated on the
5
same surface area, resulting in an increase in adsorption saturation with concentration.
6
Although the overall enzyme concentration during post-manufacture addition is much higher
7
than that of pre-manufacture addition, the saturation point was reached faster during pre-
8
manufacture addition indicating a faster spreading rate. This can be directly attributed to the
9
binding affinity between neighbouring protein molecules and the competition for available
10
surface, resulting in increased saturation. As this occurs the strength of the protein- protein
11
interactions at the surface is lower than that of the bulk allowing for the displacement of
12
protein from the surface, as shown in Figure 2.
13
The results also show that nonlinear adsorption occurs more when the enzyme was added
14
directly to the polyaphron, rather than when it was added to the bulk continuous phase. This
15
nonlinearity arises from cooperativity due to protein-protein interactions occurring as the
16
initial adsorption rate increases. These interactions are increased by the large affinity for
17
adsorption arising from direct enzyme addition. Cooperative adsorption is the result of
18
clustering arising from the self-organisation of the adsorbed monomers, and the interactions
19
between those molecules and new incoming molecules from the bulk phase. One study
20
exploring cooperative adsorption of proteins onto solid surfaces found that interactions
21
between adsorbed proteins results in minute changes in density, evolving into persistent
22
density transitions after continued adsorption; this leads to the formation of protein patches
23
next to loosely covered surface regions [21]. Another study examined a cluster theory behind
24
protein adsorption, investigating three pathways; direct adsorption onto the surface, accretion
25
of the adsorbed monomer into the cluster, and piggyback deposition allowing for
1
incorporation of the adsorbed monomer into the cluster [22]. They found that in the absence
2
of direct adsorption the rate of adsorption will always be less than ideal due to the overall
3
decrease in available surface area. When adsorption is coupled with clustering, several
4
regimes are observed based on the rate of direct adsorption and the growth of clustering
5
resulting in cooperativity. The onset of clustering has been proven to be the direct result of
6
electrostatic interactions as well as interactions due to an increased binding affinity resulting
7
from conformational changes. For charged surfaces initial adsorption is driven by coulombic
8
interactions with the surface [23], with clustering being formed by electrostatic interactions
9
with the resulting electric field generated by pre-adsorbed proteins. However, for
10
hydrophobic surfaces initial adsorption is thought to be driven by hydrophobic interactions
11
with clustering being supported by lateral interactions between adsorbed proteins, as well as
12
increased hydrophobic affinity due to possible conformational change.
13 14
Conclusion
15
An investigation into the potential use of CLAs for enzyme immobilization was conducted
16
using both hydrophilic (aprotinin) and hydrophobic (lipase and α-chymotrypsin) enzymes.
17
Through the use of CSLM, the immobilization was predominantly achieved through surface
18
adsorption. Insights into protease degradation using α-chymotrypsin showed that lysozyme
19
affinity for the outer aqueous layer of the aphron was much greater than the inner solvent
20
core. Also, by analyzing the zeta potential of the loaded CLAs, it was found that
21
immobilization greatly decreased the surface charge of the CLA owing to enzyme adsorption
22
onto the CLA surface. Furthermore, adsorption of the charged proteins aprotinin and α-
23
chymotrypsin was found to increase when the pH was closest to their respective pIs,
24
indicating that repulsive forces between free protein molecules hindered immobilization.
25
Examination of adsorption profiles showed that hydrophobic interactions were the main force
1
contributing to adsorption. This was verified as hydrophobic enzymes, lipase and α-
2
chymotrypsin, gave the best performance. The effect of increased enzyme concentration
3
showed a direct increase in immobilization, with saturation being observed faster when the
4
enzyme was added directly to the formulation due to higher initial adsorption rates resulting
5
from increased surface affinity, higher induced protein-protein interactions and cooperativity.
6 7 8
Acknowledgments
9
The authors are grateful for the partial funding received from MC2 Biotek, Derek Wheeler,
10
Stephen Lenon and Fraser Steele from Drug Delivery Solutions for their insight, and to
11
Christine Seifried for obtaining the confocal microscope images. The microscope is located
12
in the Qatar Carbonates and Carbon Storage Research Centre and was purchased using
13
funding from Qatar Petroleum, Shell and Qatar Science and Technology Park.
14
1 2 3
List of References.
4 5
[1] S.B. Lamb, Enzyme Immobilization on Colloidal Liquid Aphrons (CLAs) and the
6
Development of a Continuous Membrane Bioreactor, Imperial College London, 1999.
7
[2] G. Bickerstaff, Immobilization of Enzymes and Cells, Humana Press Inc1997.
8
[3] F. Sebba, Foams and Biliquid foams- Aphrons, John Wiley & Sons Ltd.1987.
9
[4] G.J. Lye, M. Rosjidi, O.P. Pavlou, D.C. Stuckey, Immobilization of Candida cylindracea
10
lipase on colloidal liquid aphrons (CLAs) and development of a continuous CLA-membrane
11
reactor, Biotechnol.Bioeng., 51 (1996) 69-78.
12
[5] W. Norde, C.E. Giacomelli, Conformational changes in proteins at interfaces: from
13
solution to the interface, and back, Macromol Symp, 145 (1999) 125-136.
14
[6] M. Kleijn, W. Norde, The adsorption of proteins from aqueous solution on solid surfaces,
15
Heterogen Chem Rev, 2 (1995) 157-172.
16
[7] N. Miletic, V. Abetz, K. Ebert, K. Loos, Immobilization of Candida antarctica lipase B on
17
Polystyrene Nanoparticles, Macromol Rapid Comm, 31 (2010) 71-74.
18
[8] W. Norde, C.E. Giacomelli, BSA structural changes during homomolecular exchange
19
between the adsorbed and the dissolved states, J Biotechnol, 79 (2000) 259-268.
20
[9] W. Norde, F. Macritchie, G. Nowicka, J. Lyklema, Protein Adsorption at Solid Liquid
21
Interfaces - Reversibility and Conformation Aspects, J.Colloid Interface Sci., 112 (1986) 447.
22
[10] M. Wahlgren, T. Arnebrant, Protein Adsorption to Solid-Surfaces, Trends Biotechnol, 9
23
(1991) 201-208.
24
[11] D.T.H. Wassell, G. Embery, Adsorption of Bovine Serum-Albumin to Titanium Powder,
25
J Dent Res, 74 (1995) 836-836.
1
[12] S.B. Lamb, D.C. Stuckey, Enzyme immobilization on colloidal liquid aphrons (CLAs):
2
the influence of system parameters on activity, Enzyme Microb.Technol., 26 (2000) 574-581.
3
[13] S.B. Lamb, D.C. Stuckey, Enzyme immobilisation on colloidal liquid aphrons (CLAs):
4
the influence of protein properties, Enzyme Microb.Technol., 24 (1999) 541-548.
5
[14] G.J. Lye, Stereoselective Hydrolysis of DL-Phenylalanine methyl ester and Separation
6
of L-Phenylalanine
7
Techniques, 11 (1997) 611-616.
8
[15] G.J. Lye, D.C. Stuckey, Structure and stability of colloidal liquid aphrons, Colloids and
9
surfaces.A, Physicochemical and engineering aspects, 131 (1998) 119-136.
using
aphron
immobilised
alpha-chymotrypsin,
Biotechnology
10
[16] D. Chakravorty, S. Parameswaran, V.K. Dubey, S. Patra, Unraveling the Rationale
11
Behind Organic Solvent Stability of Lipases, Appl Biochem Biotech, 167 (2012) 439-461.
12
[17] V.B. Junyaprasert, V. Teeranachaideekul, T. Supaperm, Effect of Charged and Non-
13
ionic Membrane Additives on Physicochemical Properties and Stability of Niosomes, Aaps
14
Pharmscitech, 9 (2008) 851-859.
15
[18] H. Sis, M. Birinci, Effect of nonionic and ionic surfactants on zeta potential and
16
dispersion properties of carbon black powders, Colloid Surface A, 341 (2009) 60-67.
17
[19] M. Vasudevan, J.M. Wiencek, Mechanism of the extraction of proteins into Tween 85
18
nonionic microemulsions, Ind Eng Chem Res, 35 (1996) 1085-1089.
19
[20] M. van der Veen, M.C. Stuart, W. Norde, Spreading of proteins and its effect on
20
adsorption and desorption kinetics, Colloid Surface B, 54 (2007) 136-142.
21
[21] M. Rabe, D. Verdes, S. Seeger, Understanding Cooperative Protein Adsorption Events at
22
the Microscopic Scale: A Comparison between Experimental Data and Monte Carlo
23
Simulations, J Phys Chem B, 114 (2010) 5862-5869.
24
[22] A.P. Minton, Effects of excluded surface area and adsorbate clustering on surface
25
adsorption of proteins. II. Kinetic models, Biophys.J., 80 (2001) 1641-1648.
1
[23] A.L. Creagh, J.M. Prausnitz, H.W. Blanch, Structural and Catalytic Properties of
2
Enzymes in Reverse Micelles, Enzyme Microb.Technol., 15 (1993) 383-392.
3
[24] K. Gekko, Y. Hasegawa, Compressibility Structure Relationship of Globular-Proteins,
4
Biochemistry (N.Y.), 25 (1986) 6563-6571.
5 6
1 2
List of Figures
3 4
Figure 1: Lysozyme-FITC fluorescence images of polyaphron manufactured at PVR 4. A)
5
Before polyaphron dispersion, B) After polyaphron dispersion, C) After proteolysis.
6
Excitation wavelength 488nm; magnification x63, laser intensity 4.5 %.
7
Figure 2: Adsorption profile for pre-manufacture enzyme addition as function of increasing
8
enzyme concentration at PVR 4 and 22°C. A) pH 4.8; B) pH =7.5 (Lipase-6.2), and C)
9
pH=8.5. Error bars are reported SD, n=4.
10
Figure 3: Adsorption profile for post-manufacture enzyme addition as function of increasing
11
enzyme concentration at PVR 4 and 22°C. A) pH 4.8; B) pH =7.5 (Lipase-6.2), and C)
12
pH=8.5. Error bars are reported SD, n=4.
13
Figure 4: Zeta potential difference recorded after enzyme immobilization over the pH range
14
4-9. Analysis carried out at ambient temperature 22°C. Error bars are reported SD, n=3.
15 16
1 2
List of Tables
3 4 5 6 7 8 9 10 11 12 13 14 15 16
Table 1: Enzyme properties a
Enzyme
pI
Molecular Weight[Da]
Lipase
4.1
45000
Aprotinin
10.5
6512
910 -
α-Chymotrypsin
8.75
25000
908
Hydrophobicity [cal/residue] b
a-Total hydrophobicity [24] b- Estimated hydrophobicity [13] 17 18 19
Table 2: Average zeta potential measurements taken before and after enzyme immobilization
20
(Error: SD, n=3) Formulation
Zeta Potential (mV) pH 4.8 pH 6.2/7.5 Before Immobilization
pH 8.6
Blank (phosphate)
-19.31±0.96
-23.96±1.91
-24.95±1.24
Blank (Tris HCl)
-10.61±0.98
-17.17±0.96
-20.62±1.15
After Immobilization
21 22 23
Lipase
-6.97±0.65
-12.46±0.95
-16.17±1.01
Aprotinin
-12.36±0.84
-18.57±1.02
-23.40±1.81
α-Chymotrypsin
-9.20±0.69
-23.45±1.55
-23.63±0.98
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Figure 3
1 2 3
4
Figure 4