Luís Gustavo Godinho Barreira
Mestre em Parasitolgia Médica
CHARACTERIZATION OF SOL-GEL MATRICES
WITH ENTRAPPED CUTINASE
Dissertação para obtenção do Grau de Doutor em
Engenharia Química e Bioquímica
Orientador: Susana Filipe Barreiros, Prof. Associada com
Agregação, Faculdade de Ciências e Tecnologia.
Co-orientadores: Eurico J. Cabrita, Prof. Auxiliar, Faculdade
de Ciências e Tecnologia; e João Carlos Lima, Prof Auxiliar,
Faculdade de Ciências e Tecnologia
Março de 2012
“Science should be as simple as
possible but not simpler”:
Albert Einstein
2
Aknowledgments
To Prof. Susana Barreiros, my advisor during all four year of research, I am much
obliged for her accurate scientific advice and permanent availability in guiding the
work described in this thesis.
I thank Prof. João Lima and Prof. Eurico Cabrita for their tutoring and collaboration
that made fluorescence and NMR studies possible and always gave me wise scientific
advice.
To Dr. Alexandre Babo and Ms. Rita Rodrigues, my very thanks for their availability
in collaborating in the supercritical drying of the sol-gel matrices.
Thanks to Dr. Pedro Vidinha, who always gave his opinion about ongoing works and
shared his knowledge and previous expericence.
I also thank Fundação para a Ciência e a Tecnologia for financial support that allowed
me to do this thesis.
To all of you who, one way or another, helped me carry through this project,
My very best regards.
iii
Resumo
Imobilizou-se a enzima cutinase, produzida por Fusarium solani pisi, em matrizes de sol-gel
de composição 1:5 tetrametoxisilano (TMOS)/n-butiltrimetoxisilano (BTMS). Mediu-se a
actividade específica da enzima em função da carga da mesma nas matrizes que variou entre
cerca de 0.1 % e 7 %. No sentido de compreender o aumento pronunciado de actividade
específica que se observou à medida que a carga de enzima nas matrizes diminuía, recorreuse à titulação de centros activos, baseada na inactivação da enzima pelo inibidor paraoxon.
Verificou-se que o número de centros activos da enzima que estavam disponíveis diminuía
com o aumento da carga de enzima nas matrizes, o que sugere a ocorrência de agregação das
moléculas de enzima nas matrizes mais carregadas. Para confirmar a ocorrência deste
fenómeno, utilizou-se a espectroscopia de fluorescência baseada na resposta do único
resíduo de triptofano da cutinase. As medições do decaimento da anisotropia de
fluorescência conduziram a parâmetros de ordem de baixa magnitude, o que indica que as
restrições impostas ao triptofano pelo seu microambiente são poucas. Esta situação é
consistente com o facto de o triptofano estar apenas parcialmente exposto na molécula de
cutinase. Verificou-se ainda que o parâmetro de ordem aumentava com o aumento da carga
de enzima nas matrizes, o que indica perda de mobilidade do resíduo de triptofano nas
matrizes de sol-gel mais carregadas e é consistente com a hipótese de agregação da enzima.
O efeito da carga da enzima na mobilidade do solvente dentro da matriz foi aferido mediante
a determinação do coeficiente de difusão translacional do solvente, utilizando espectroscopia
de ressonância magnética nuclear de alta resolução com rotação no ângulo mágico e pulsos
de gradiente de campo (HR-MAS PFGSE NMR). Os coeficientes de difusão obtidos indicam
que a carga da enzima nas matrizes tem um efeito desprezável na mobilidade do solvente.
Estudou-se também a estabilidade térmica da cutinase imobilizada nas matrizes de sol-gel de
composição 1:5 TMOS/BTMS, numa gama de cargas de enzima entre cerca de 0.1 % e 4 %.
Verificou-se que a incubação das preparações de enzima imobilizada a 40 ºC durante 24
horas tinha um efeito positivo na actividade específica da enzima, contrariamente ao
constatado para incubação a temperaturas mais elevadas e para o mesmo tempo de
exposição. O impacto da temperatura na actividade específica da enzima foi mais
pronunciado nas matrizes com menor carga proteica. No entanto, após tratamento a 100 ºC, a
actividade específica da enzima imobilizada atingiu valores muito semelhantes em todas as
matrizes, independentemente da carga de enzima respectiva. Os espectros obtidos por
iv
espectroscopia de fluorescência de estado estacionário não revelaram aumento da emissão de
fluorescência como resultado da exposição à temperatura, e a espectroscopia de
fluorescência com resolução temporal mostrou que os tempos do decaimento de
fluorescência do triptofano da cutinase não aumentavam com o aumento da temperatura de
incubação das matrizes com enzima. Estes dois factos indicam que, contrariamente ao que
acontece com a cutinase em solução, a cutinase imobilizada e submetida a temperaturas
elevadas não sofre um processo de desnaturação caracterizado por uma elevada mobilidade
conformacional na região do resíduo de triptofano. Este efeito de protecção não é mediado
pelo grau de empacotamento da enzima, já demonstrado para cargas de enzima mais
elevadas, uma vez que nem as matrizes com menos enzima apresentaram efeitos de
desnaturação. As matrizes são hidrofóbicas e têm um baixo teor de água. Um certo grau de
desidratação da enzima como resultado da exposição a temperaturas moderadas poderá ser
benéfico para a mobilidade conformacional da enzima e, consequentemente, para a
actividade da mesma, como se constatou neste estudo, mas passar a ter um efeito negativo no
funcionamento da enzima para valores de temperatura mais elevados. Isto poderia explicar a
semelhança dos valores da actividade específica da enzima após exposição às temperaturas
mais elevadas utilizadas no presente estudo, independentemente da carga enzimática nos
vários suportes testados.
Mediu-se ainda a migração, nas matrizes de sol-gel de composição 1:5 TMOS/BTMS, do
composto 2-fenil-1-propanol, um dos substratos da reacção de monitorização da actividade
da cutinase em meios não aquosos, bem como da água dissolvida em diferentes solventes
(acetonitrilo a dois valores de actividade da água, n-hexano e metanol com um dado teor em
água), e mediu-se ainda a migração dos próprios solventes, com vista a uma melhor
compreensão do processo global de catálise pela enzima imobilizada. A migração das
espécies consideradas foi quantificada através dos coeficientes de difusão translacional
respectivos, determinados por espectroscopia HR-MAS PFGSE NMR. Os coeficientes de
difusão dos solventes foram também determinados na ausência de matriz, no sentido de dar
conta das diferenças de viscosidade dos solventes. Os resultados obtidos mostram que a
difusão do solvente através da matriz de sol-gel é altamente influenciada pela natureza
química do solvente e pela sua interacção com a matriz. Para racionalizar os resultados
experimentais obtidos e o processo de transferência de massa nas matrizes de sol-gel,
propõe-se um modelo semelhante ao que é utilizado em cromatografia, envolvendo permuta
de moléculas entre diferentes domínios difusionais (difusão nos poros e difusão à superfície).
v
Abstract
Cutinase from Fusarium solani pisi was entrapped in sol-gel matrices of composition 1:5
tetramethoxysilane:n-butyltrimethoxysilane (TMOS/BTMS), and its specific activity was
measured as a function of enzyme loading in the range of ca. 0.1 % to 7 %. To elucidate the
pronounced increase in specific activity that was observed as enzyme loading decreased, an
active site titration technique was applied, based on enzyme inactivation by the inhibitor
paraoxon. It was found that the number of available enzyme activate sites decreased as
enzyme loading in the matrices increased, suggesting that enzyme aggregation occurred in
the more heavily loaded sol-gel supports. The impact of enzyme loading on the packing of
cutinase inside the matrices was studied by fluorescence spectroscopy based on the single
tryptophan residue of cutinase. Fluorescence anisotropy decay measurements led to
relatively low order parameters, indicating that the restrictions imposed on tryptophan by its
microenvironment are small, which is consistent with the fact that tryptophan is only
partially exposed on the cutinase molecule. The order parameter increased as enzyme
loading increased, indicating loss of mobility of the tryptophan in the more heavily loaded
sol-gel matrices, which is consistent with enzyme aggregation. The effect of enzyme loading
on solvent mobility within the matrix was assessed by determining the self-diffusion
coefficient of the solvent using High Resolution Magic Angle Spinning (HR-MAS) Pulsed
Field Gradient Spin Echo (PFGSE) NMR spectroscopy. The diffusion coefficients
determined indicate that enzyme loading has a negligible effect on solvent mobility.
We also studied the thermal stability of cutinase entrapped in 1:5 TMOS/BTMS sol-gel
matrices, at enzyme loadings in the range of ca. 0.1 % to 4 %. We found that submitting the
entrapped enzyme to 40 ºC for 24 h had a positive effect on enzyme specific activity and that
thermal treatment for the same length of time at higher temperatures no longer brought about
any activity enhancements. The impact of temperature on enzyme specific activity was more
pronounced in the case of more dilute matrices, whose activity nonetheless became very
similar to that of the more heavily loaded matrices after treatment at 100 ºC. Steady-state
fluorescence measurements did not reveal an increase in fluorescence emission with
exposure to temperature, and time resolved fluorescence showed that the fluorescence decay
times of the single tryptophan of cutinase did not increase as the temperature of incubation of
the sol-gel matrices increased. Both findings indicate that unlike what happens with cutinase
vi
in solution, entrapped cutinase submitted to higher temperatures does not suffer a
denaturation process characterized by ample conformational mobility in the region of the
tryptophan residue. This protective effect is not mediated by enzyme packing, known to
occur at higher enzyme loadings, because not even the more dilute matrices showed
evidence of enzyme denaturation. The matrices are hydrophobic and have little water. A
certain extent of enzyme dehydration upon thermal treatment at moderate temperatures could
be beneficial for enzyme conformational mobility and hence enzyme activity, as it was also
observed in this study, and become detrimental to enzyme function at higher temperatures.
This would explain the similarity of enzyme specific activity values after thermal treatment
at the highest temperature tested, irrespective of enzyme loading.
Additionally, we measured the displacement of the species 2-phenyl-1-propanol, used to
monitor the activity of cutinase in nonaqueous media, and of water dissolved in different
solvents (acetonitrile at two values of water activity, n-hexane and methanol with a given
water content), as well as the displacement of the solvents themselves, in 1:5 TMOS/BTMS
sol-gel matrices, to elucidate the overall process of catalysis by the entrapped enzyme. The
molecular displacement was measured in terms of the self-diffusion coefficient, as
determined by HR-MAS PFGSE NMR spectroscopy measurements. The solvent selfdiffusion was also determined in the absence of the matrix, in order to account for
differences in solvent viscosity. Our results show that the diffusion of the solvent through the
sol-gel matrix is highly influenced by the chemical nature of the solvent and its interactions
with the sol-gel matrix. A model similar to that used in chromatography, involving molecular
exchange between different diffusion domains (pore diffusion and surface diffusion) is
proposed to rationalize the experimental results and the mass transfer process in sol-gel
matrices.
vii
Nomenclature
TMOS – Tetramethoxysilane
BTMS - n-butyltrimethoxysilane
Km – Michaelis–Menten constant
kcat – turnover number
T – Temperature
PFGSE HRMAS NMR - pulsed field gradient spin-echo high resolution magic angle
spectroscopy nuclear magnetic resonance
NaF – sodium fluoride
CO2 - carbon dioxide
PVA – polyvinyl alcohol
NaOH - sodium hydroxide
2F1P - (R,S)-2-phenyl-1-propanol
GC – Gas Chromatography.
Paraoxon - diethyl-p-nitrophenyl phosphate.
pNPB – p-nitrophenyl butyrate
D - diffusion coefficient
Dav - average diffusion coefficient
Dslow – slow diffusion coefficient
Dfast – fast diffusion coefficient
ACN - acetonitrile
viii
aW – water activity
MeOH – methanol
ix
General Index
Chapter 1 ...............................................................................................................................1
Introduction and aims of the thesis ........................................................................................1
1.1. Sol-gel encapsulation. .................................................................................................1
1.2. Scope of the thesis. Focus on sol-gel entrapped cutinase. ..........................................8
1.3. Structure of the thesis ...............................................................................................15
1.4. References .................................................................................................................17
Chapter 2 .............................................................................................................................24
Assessing the aggregation of cutinase in sol-gel matrices ..................................................24
2.1. Abstract .....................................................................................................................24
2.2. Introduction ...............................................................................................................25
2.3. Materials and methods ..............................................................................................27
2.3.1. Materials .............................................................................................................27
2.3.2. Cutinase immobilization in sol-gel ....................................................................27
2.3.3. Enzyme activity assays in transesterification reactions .....................................28
2.3.4. Reutilization assays ............................................................................................28
2.3.5. Enzyme particle sizes .........................................................................................29
2.3.6. Transesterification reaction analysis ..................................................................29
2.3.7. Free enzyme inhibition assays............................................................................29
2.3.8. Immobilized enzyme inhibition assays ..............................................................30
2.3.9. Fluorescence anisotropy decays .........................................................................30
2.3.10. HR-MAS NMR diffusion spectroscopy ...........................................................32
2.4. Results and Discussion .............................................................................................33
2.5. Conclusions ...............................................................................................................40
2.6. Acknowledgments .................................................................................................41
2.7. References .................................................................................................................41
Chapter 3 .............................................................................................................................48
Thermal stability of sol-gel entrapped cutinase ...................................................................48
3.1. Abstract .....................................................................................................................48
3.2. Introduction ...............................................................................................................49
3.3. Materials and methods ..............................................................................................51
x
3.3.1. Materials .............................................................................................................51
3.3.2. Cutinase immobilization in sol-gel ....................................................................51
3.3.3. Thermal treatment ..............................................................................................52
3.3.4. Enzyme activity assays.......................................................................................52
3.3.5. Transesterification reaction analysis ..................................................................52
3.3.6. Fluorescence anisotropy decays .........................................................................53
3.4. Results and Discussion .............................................................................................53
3.5. Conclusions ...............................................................................................................59
3.6. Acknowledgments .................................................................................................59
3.7. References .................................................................................................................60
Chapter 4 .............................................................................................................................63
Solvent mobility in sol-gel matrices as measured by HR-MAS PFGSE NMR spectroscopy
.............................................................................................................................................63
4.1. Abstract .....................................................................................................................63
4.2. Introduction ...............................................................................................................64
4.2.1. NMR Theory ......................................................................................................65
4.2.2. HR-MAS NMR diffusion spectroscopy .............................................................66
4.2.3. Restricted diffusion ............................................................................................66
4.3. Materials and methods ..............................................................................................67
4.3.1. Materials .............................................................................................................67
4.3.2. Sol-gel assay preparation ...................................................................................67
4.3.3. NMR assays........................................................................................................68
4.4. Results and Discussion .............................................................................................69
4.4.1. Solution self-diffusion coefficients in the absence of matrix .............................70
4.4.2. HRMAS PFGSE NMR self-diffusion coefficients ............................................72
4.4.3. Solvent composition affects exchange regime between diffusion domains .......75
4.4.4. Model for the intermediate regime .....................................................................75
4.5. Conclusions ...............................................................................................................78
4.6. Acknowledgments .................................................................................................78
4.7. References .................................................................................................................79
5. Conclusions .....................................................................................................................83
5.1. Reference ..................................................................................................................86
xi
Figures Index
Figure 1.1.: Structural representations of an alkoxide, tetramethoxysilane (TMOS) - A
and an alkoxysilane, n-butyltrimethoxysilane (BTMS) - B ................................................ 2
Figure 1.2.: Effect of enzyme loading on the activity of sol-gel entrapped Pseudomona
cepacia lipase (from Reetz et al. 1996 ref 64). ........................................................................ 8
Figure 1.3. : (A) 3D backbone fold of Fusarium solani pisi cutinase, showing
accessibility of the active site region. The dynamics of the backbone is also indicated. Red,
mobile blue, rigid (from Egmond and Vlieg61) (B) Ribbon representation of Fusarium
solani pisi cutinase, highlighting two of the four cysteins (Cys-31 Cys-109) in the
neighborhood of the buried single tryptophan residue (Trp-69) (from Vidinha et al.86). .... 9
Figure 1.4. : Schematic view of an entrapped enzyme with a few water molecules inside a
sol-gel pore (from Frenkel-Mullerad and Avnir70). ............................................................ 11
Figure 2.1. : Impact of enzyme loading of sol-gel matrix on cutinase specific activity.
Five matrices with enzyme loadings of 0.08 %, 0.27 %, 0.52 %, 0.97 % and 3.65 % were
prepared and used six times consecutively in a transesterification reaction performed in nhexane at room temperature, yielding the six data sets shown in the figure., 1st , 2nd ▲,
3rd △, 4th ●, 5th ○, 6th. The lines are trend-lines. The standard deviations of enzyme loading
values range from ca. 17 % for higher enzyme loadings, to ca. 32 % for the two lowest
enzyme loadings. ................................................................................................................ 33
Figure 2.2. : Residual activity of free cutinase in aqueous buffer after exposure to varying
concentrations of the inhibitor paraoxon. Enzyme activity was measured by adding pNPB
and measuring the release of p-nitrophenol at λ = 405 nm. [cutinase] = 4.2E-06 M. The
line is a trend-line. .............................................................................................................. 35
Figure 2.3. : Residual specific activity of sol-gel entrapped cutinase after exposure, in
acetonitrile, to concentrations of the inhibitor paraoxon setting a 1:10 enzyme:inhibitor
molar ratio. Enzyme activity was measured in a transesterification reaction performed in
n-hexane at room temperature. Five matrices with enzyme loadings of 0.05 %, 0.10 %,
0.47 %, 1.03 %, 2.50 % and 6.88 % were used. The line is a trend-line. Standard
deviations of enzyme loading values as in Figure 1. .......................................................... 37
Figure 2.4. : Time resolved anisotropy decay curve, r(t), of the single tryptophan residue
of cutinase entrapped in sol-gel matrices, and the corresponding residual of the fitting by
equation 3. A) Matrix with 3.63 % enzyme loading B) Matrix with 0.06 % enzyme
loading. Standard deviations of enzyme loading values as in Figure 1. ............................. 38
xii
Figure 3.1. : Initial rates of sol-gel entrapped cutinase kept at room temperature (blank)
and submitted to a number of temperature cycles (initial and final temperatures of 22 ºC
and 80 ºC, 5 ºC increases and holding for 10 min at each temperature plateau). Enzyme
loading: gray bars, 1.38 % white bars, 0.52 %. Enzyme specific activity was measured in a
transesterification reaction performed in n-hexane at room temperature. The standard
deviations of enzyme loading values are of ca. 17 %. ........................................................ 54
Figure 3.2. : Impact of the incubation of enzyme loaded sol-gel matrices at temperatures
from 40 to 100 ºC, for 24 h, on enzyme specific activity. Enzyme loading: ,0.08 % ,
0.27 % ▲, 0.52 % △, 0.97 % , 3.65%. The data points at 20 ºC represent supports kept
at room temperature. Enzyme activity was measured in a transesterification reaction
performed in n-hexane at room temperature. The standard deviations of enzyme loading
values range from ca. 17 % for higher enzyme loadings, to ca. 32 % for the two lowest
enzyme loadings. ................................................................................................................ 55
Figure 3.3. : Decay times of cutinase entrapped in sol-gel matrices, obtained from the
fitting of the fluorescence decay curves by sums of three exponentials. Enzyme loading:
,0.06 % ○, 0.49 % □, 1.6%, , 3.63 %.The scatter observed for the lowest enzyme
loading results from photobleaching due to the higher measurement times under laser
irradiation. The standard deviations of enzyme loading values are of ca. 17 %, except for
the lowest enzyme loading, where it is of ca. 32 %............................................................ 57
Figure 5.1. : N84W cutinase mutant, with additional tryptophan residue shown as sticks
(on top). Also shown as sticks are the residues of the catalytic triad (Ser120, Asp175,
H188). ................................................................................................................................. 85
Scheme 1: Nucleophilic chemical attack on the Si atom (from Pierre, 2004) ..................... 3
Scheme 2: Representation of the diffusion of a substrate (S) molecule in a heterogeneous
system under conditions of two-site exchange. Limiting diffusion environments are
represented by DP –diffusion in the pore and DS –diffusion in the surface; k1 and k-1
represent the exchange rates between the two domains. .................................................... 77
xiii
Tables Index
Table 1.1. : Lipase loadings and relative amounts of immobilized enzyme in sol-gel
matrices. .................................................................................................................................7
Figure 2.2. : Residual activity of free cutinase in aqueous buffer after exposure to varying
concentrations of the inhibitor paraoxon. Enzyme activity was measured by adding pNPB
and measuring the release of p-nitrophenol at λ = 405 nm. [cutinase] = 4.2E-06 M. The
line is a trend-line. ...............................................................................................................35
Table 2.1. : Impact of enzyme loading of sol-gel matrix on cutinase specific activity. The
two matrices were assayed in acetonitrile, in the hydrolysis of pNPB at room temperature.
Standard deviations of enzyme loading values as in Figure 1 .............................................36
Table 2.3. : Self-diffusion coefficients determined by HR-MAS PFGSE diffusion NMR 40
Table 3.1. : Average increase in enzyme specific activity after incubation of the enzyme
loaded sol-gel matrices for 24 h at 40 ºC, relative to incubation for 24 h at 100 ºC. Enzyme
activity was measured in a transesterification reaction performed in n-hexane at room
temperature. .........................................................................................................................55
Table 4.1. : Self-diffusion coefficient determined by PFGSE NMR technique of water, 2phenyl-1-propanol (2F1P), acetonitrile (ACN), methanol (MeOH) and n-hexane in the
different reference solutions. Literature values are for the pure solvents, except for
methanol ..............................................................................................................................71
Table 4.2. : Self-diffusion coefficient determined by PFGSE NMR technique of water, 2phenyl-1-propanol (2F1P), acetonitrile (ACN), methanol (MeOH) and n-hexane in the
different reference solutions. Literature values are for the pure solvents, except for
methanol. .............................................................................................................................74
xiv
Chapter 1
Introduction and aims of the thesis
1.1. Sol-gel encapsulation.
Enzymes are abundantly found in physiological mechanisms as they are very effective and
precise (bio)catalysts that perform and regulate processes in living matter. The potential of
enzymes is still far from being fully exploited. Indeed estimates generally agree that less than
1% of microorganisms in the environment have been cultivated to date and their enzymes
identified1. Although many enzymes remain to be discovered, a vast number that catalyse a
huge array of reactions have been identified and characterized. Their practical use has been
realized within various industrial processes and products, from laundry detergents to finechemicals, pharmaceuticals, biosensors, bioremediation, biobleaching, polymerase chain
reaction, protein digestion in proteomic analysis, and biofuel cells2.
The industrial use of enzymes requires specific approaches to overcome limited long-term
stability, activity problems, difficulties in separating the products from the enzyme, and
hurdles arising upon reusing the biocatalyst. In order to solve the problems of stability and
recyclability, several approaches as membrane reactors, cross-linked crystalline enzymes,
two-phase systems or micro-emulsions and immobilization, have been applied3.
A highly attractive feature of immobilization would obviously be an increase in stability and
catalytic activity, key properties for industrial applications, in addition to selectivity.
Immobilization of an enzyme usually entails the interaction of two species, the enzyme and
the carrier, the surface properties of both being important in this respect. There has been
consensus that one of the best means to minimize the influence of the carrier on the structure
of an enzyme is to encapsulate it. The sol-gel method appears to be the most widely
employed technique4 and has been used for enzyme entrapment in such diverse fields and
1
applications as biomedical products5, products for the treatment of intolerance to lactose6,
artificial
organs
bioremediation11;12;13,
design7,
chemical
antibiotics
production8;9,
sensors14;15,
biosensors16;17;18;19,
biomass
hydrolysis10,
including
specific
glucose20;21 and pesticide sensors22. Sol-gel matrices are highly porous silica materials whose
synthesis is recognized as relatively benign for most enzymes.
In a typical sol-gel synthesis protocol, the precursors used are alkoxides of the type Si(OR)4,
or alkoxysilanes of the type XSi(OR)3 or XX’Si(OR)2, in which X and X’ designate organic
groups, directly linked to the Si atom by a Si-C bond at one end, and bearing various
functionalities at the other end. In the alkoxides, R is often a methyl group, so that the
precursor is termed tetramethoxysilane, or TMOS (Figure 1.1).
A
B
Figure 1.1.: Structural representations of an alkoxide, tetramethoxysilane (TMOS) – A, and
an alkoxysilane, n-butyltrimethoxysilane (BTMS) – B.
The first sol-gel reactions to which an alkoxide precursor is submitted are of the hydrolysis
type23, leading to the replacement of OR ligands by OH ones.
Si(OR)4 + H2O  Si(OR)3 (OH) + ROH
(e.g.) Si(OCH3)4 + H2O  Si(OCH3)3 (OH) + CH3OH
Hydrolysis is then followed by condensation reactions.
Si(OR)3 (OH) + Si(OR)3 (OH)  (RO)3SiOSi(OR)3 + H2O
(e.g.) Si(OCH3)3 (OH) + Si(OCH3)3 (OH)  (H3CO)3SiOSi(OCH3)3 + H2O
2
With Si-O-based precursors, the mechanism of hydrolysis reactions depends on nucleophilic
chemical attack on the Si atom (Scheme 1), which in turn depends on the partial positive
electronic charge δ+ carried by this atom.
Scheme 1: Nucleophilic chemical attack on the Si atom (from Pierre, 2004).
Hence, the nucleophilic attack of O atoms from water, which carries a partial negative charge
δ-, is not as easy as in transition metal alkoxides, meaning that both the hydrolysis and
condensation reactions in the latter cases are fast24, so that it becomes difficult to separate
them experimentally.
On the other hand, as the hydrolysis and condensation reactions of Si alkoxides are slow,
they need to be catalyzed, either by acids (e.g. H+), which carry a strong positive charge and
are able to attack the O (δ-) atoms from the alkoxy OR groups, or by bases, which carry
strong negative charges (e.g. OH-, but also strong Lewis bases, such as F- ions).
This can be an advantage because the hydrolysis and condensation rates can be controlled.
Overall, silica gels with a texture closer to polymeric gels are obtained when the hydrolysis
rate is faster than the condensation rate, which requires adding an acid catalyst or proton
donor25. Proton acceptors, i.e. bases, accelerate the condensation reactions more than
hydrolysis, which favors the formation of denser colloidal silica particles. Therefore when
the condensation continues, the gel is formed and the interstitial spaces of this gel are filled
with water and alcohol; hence the designation ‘hydrogel’. The ability to control this kinetics
has important consequences regarding the adaptation of silica chemistry to further enzyme
encapsulation.
If only simple silica alkoxides Si(OR)4 are used as precursors, the wet gels mostly carry SiOH sides groups which are hydrophilic and induce considerable capillary contraction
(typically 70 %) of the whole structure. On the other hand, in 100% alkoxysilane gels, the
3
pore surfaces carry hydrophilic silanols (Si-OH) in between the hydrophobic Si-X ones, a
situation which bears some similarity to the protein surface of an enzyme. However an
alkoxysilane XSi(OR)3 carrying hydrophobic groups X is often more difficult to hydrolyse
and condense than the alkoxides26,
because the organometallic Si-X bond cannot be
hydrolyzed.
Consequently, if mixtures of alkoxide and alkoxyalkyl are used, the gel network is initially
built mostly by the alkoxide, while the alkoxyalkyl mostly covers the pore surface in a
second stage reaction. In the xerogels obtained after solvent evaporation, the capillary
stresses are largely attenuated by hydrophobic groups ending up more densely distributed on
the surface of the pores27. Thus provided the proportion of alkysiloxane in the precursor
mixture is sufficiently high, the proportion of surface hydrophobic groups in the pores will
be sufficient to give a hydrophobic character to the gels. Polar liquids such as water or
aliphatic alcohols do not wet the surfaces covered by such hydrophobic groups. Sol-gel
matrices such as those made by Reetz and co-workers28;
29;30
with a high proportion of
methyl trimethoxysilane (MTMS) or other alkoxysilanes are of this type.
Supercritical drying makes it possible to avoid gel shrinkage. It consists of bringing the
liquid in the wet gel beyond its critical point. The critical temperatures of water (374 ºC) or
alcohols (ethanol 243 ºC) are too high for enzymes. However a liquid such as CO2 has a
critical temperature of 31 ºC, and can be conveniently applied to dry gels with encapsulated
proteins, resulting in matrices with enormous porosities named ‘aerogels’31. Nevertheless the
liquid in the wet gel must be exchanged for liquid CO2, which requires dialysis: water or
methanol are not miscible with liquid CO2, hence they must first be exchanged with an
intermediate liquid such as acetone or amyl acetate25. This will be a progressive and lengthy
procedure if shrinkage needs to be completely avoided.
As a further improvement it is possible to encapsulate, together with the enzyme, additives
which experiments have shown to be beneficial for the stability or bioactivity of the
biocatalyst. These additives can be hydrophobic moieties brought by alkoxysilane as
additional precursors, carrying for instance alkyl-R groups, as previously mentioned. An
important contribution was brought by Reetz and co-workers, who showed such hydrophobic
moieties could improve the activity of lipases beyond that of free enzymes using polymer
additives such as polyvinyl alcohol, or glycerol28; 30. The addition of polymers to the silica
gels provides protection of the enzymes from denaturation effects during the formation of the
4
silica matrix28, increase the gel pore size, which results in improved substrate delivery and
hence enzyme activity, and makes it possible to modify the enzyme-silica interactions
responsible for restricting the molecular motion of the entrapped enzymes32, bringing about
an increase in enzyme thermal stability.
In addition to the precursors and additives, the amount of water used to hydrolyse the
precursors can influence the activity of an encapsulated enzyme33. In cases where the amount
of water added for hydrolysis of the silica precursors was low, it was suggested that enzyme
conformation was frozen during gelation, a suggestion consistent for instance with the
observation that the activity of sol-gel encapsulated β-galactosidase was much higher in wet
gels than in dry ones.
On the other hand, sol-gel encapsulation has been known to slow down the unfolding of
proteins34, which improved their thermal stability35. A link between conformational rigidity
and enhanced thermal stability has thus been inferred36, and supporting evidence was
obtained from encapsulated creatine kinase37, in which spectroscopic analysis of the
unfolding of that enzyme showed that encapsulation in silica resulted in incomplete enzyme
denaturation at temperatures up to 90 ºC, while in solution the midpoint temperature of the
unfolding transition37 was 75 ºC.
Globally it stands out that the sol-gel network slows down any change in the enzyme
conformation, as sol-gel encapsulation consists in ‘knitting’ a porous wall, by the chemical
condensation of the silica network around the enzyme. In such process, the enzyme acts as a
template, so that its nano-capsule size is usually much larger than most of the pores which
prevail in the gel walls. By preventing large conformational changes such as large scale
unfolding, sol-gel encapsulation is likely to improve enzyme stability towards thermal or
chemical inactivation
38; 39; 40
. Nevertheless, in order to be able to transform a substrate, an
enzyme must be able to undertake the appropriate conformational changes, and hence it must
have some freedom inside the sol-gel pores. Inside the sol-gel proteins can be adsorbed or
associated with various surface functional groups, in a variety of orientations, or even
aggregated41. A fraction of the enzyme may also be in cages which are not accessible to the
substrate 42.
Therefore it can be expected that during the process of sol-gel encapsulation there may occur
some loss of activity of the immobilized enzymes, e.g. due to a smaller number of active
sites of the biocatalysts being accessible to diffusing substrate molecules, or because the
5
enzyme molecules entrapped within the sol-gel matrix exhibit lower affinity towards the
substrates, due to some level of denaturation imposed by capillary stresses upon the
polymerization of the matrix around the proteins. Another possibility is the enzyme activity
being hindered by retarded diffusion of the reactants through the porous matrix, as compared
with solution41. Limitations in diffusion rates, restricted accessibility of the entrapped protein
and/or alterations in binding constants43 are commonly reported to be behind the observed
decreases in enzyme activity upon sol-gel encapsulation.
In line with those observations, most studies on sol-gel encapsulation of enzymes have
shown that immobilization does not change the kinetic mechanism type, but only the
magnitude of kinetics constants of sol-gel encapsulated enzymes44. Usually the Michaelis
constant Km is increased, which indicates weaker substrate binding by the enzyme, while the
global kinetics measured by kcat are slower38; 45. There are exceptions though, in which kcat
has been shown to increase upon encapsulation, while Km decreased46. The best results
concern esterification reaction in organic hydrophobic solvents (i.e. isoctane) with lipases
such as that from Burkholderia cepacia 47. The activity of that lipase could be increased by a
factor up to 129, while that from Thermomyces lanuginose30 increased by a factor up to
1319.
It is conceivable in the latter case that the matrix hydrophobic moiety helps to orient the
enzyme molecules in a way that exposes their active sites to the intrapore space41, or that the
binding of the enzyme to the support is made by a region opposite to the active site48, thus
orienting the catalytic region to the organic phase. Reetz and co-workers29,30 have
experimentally observed an enhancement of lipase activity upon using TMOS combined
with an n-alkyltrimethoxysilane (n-alkylTMS) in the order methyl < ethyl < n-propyl < nbutyl. The authors showed the increased enzyme activity was due to the increased
hydrophobic character of the matrices, which in turn induced the immobilization of lipases in
an open-lid conformation, thus enhancing catalytic performance. Additionally Vidinha and
co-workers50 have shown that when cutinase was immobilized in 1:5 TMOS/n-alkylTMS
sol-gel matrices where the alkyl groups ranged from methyl to n-octyl, the recorded activity
increased up to n-butyl, and then decreased.
For industrial applications, the enzyme loading as well as the percentage of initially free
enzyme that can be retained inside the sol-gel after encapsulation are important decision
criteria (Table 1.1).
6
Table 1.1. : Lipase loadings and relative amounts of immobilized enzyme in sol-gel matrices.
Enzyme retained from
Enzyme
Enzyme loading
immobilization
solution/%
Reference
Lipase from Burkholderia cepacia
150 mg / g
60
30
Lipase from Thermomyces lanuginose
70 mg /g
60
30
Lipase from Burkholderia cepacia
15 to 60 mg /g
96
47
Lipase from Candida rugosa
< 20 mg/ g
> 95
50; 51
Lipase from Candida rugosa
62.5 mg /g
95
49
The determination of the enzyme kinetic constants is usually conducted under conditions
where the activity of the entrapped enzyme increases linearly with total enzyme content, so
as to eliminate e.g. diffusion and aggregation problems. For practical applications, enzyme
loading should be increased up to the point where the activity of the immobilized enzyme
levels off. In this respect it has been referred that while the amount of enzyme retained in the
support is slightly independent of the amount of protein in the immobilization solution, a
further increase in enzyme loading beyond a certain critical amount does not result in higher
specific catalytic activity, as compared with similar free enzyme concentrations53. Reetz and
co-workers64 have observed a decrease in the specific activity of a sol-gel entrapped enzyme
associated with the increase in enzyme loading (Figure 1.2).
Figure 1.2.: Effect of enzyme loading on the activity of sol-gel entrapped Pseudomona cepacia lipase (from
Reetz et al. 1996 ref 64).
7
Reetz and co-workers attributed this effect either to the overloading of the matrix with the
enzyme molecules, causing aggregation phenomena of the entrapped enzyme molecules
resulting in a lower degree of dispersion in the so-gel matrix, or to diffusional limitations.
1.2. Scope of the thesis. Focus on sol-gel entrapped cutinase.
Cutinases are enzymes produced by several phytopathogenic fungi that are able to hydrolyse
ester bonds in cutin, an insoluble matrix composed by lipid polyesters that covers the higher
plants54. Cutinases show also hydrolytic activity on triacylglycerols, as efficiently as
pancreatic lipases55. Cutinase from Fusarium solani pisi (Figure 1.3.) is a 22 kDa compact
one domain molecule, and its three-dimensional structure was solved to 1.0 Å. It is an α/β
protein comprising 197 residues, with a hydrophobic core comprising a slightly twisted five
parallel-stranded β-sheets surrounded by four α-helices. It is an enzyme that belongs to the
class of serine esterases and to the super-family of α/β hydrolases56. The α/β-hydrolase
pattern fold and the catalytic machinery composed by a nucleophile, an acid and a histidine,
seem to be a common feature to esterases and lipases. Thus the active site consists of a
catalytic triad – Ser-120, Asp-175 and His-188 – and unlike most lipases, the catalytic serine
is not buried under an amphipatic loop, but is accessible to the solvent: it is located at one
extreme of the protein ellipsoid, at the bottom of a crevice, the oxyanion hole57, delimited by
two loops (residues 80-87 and 180-188). Additionally a comparison between the structures
of native cutinase and of a covalently inhibited complex with n-diethyl-p-nitrophenyl
phosphate, revealed a preformed oxyanion cavity58. The catalytic centre is thus directly
exposed to the solvent, suggesting no need for structural rearrangements for substrate
binding, contrarily to lipases where lid opening is imperative59.
The oxyanion delimits two loops which are flexible, bear hydrophobic amino acids that
constitute the lipid binding site of cutinase, and provide dynamic behavior at the crevice
entrance surface. On the other hand, the oxyanion hole provides the electrophylic
environment needed to stabilize the negative charges produced in the nucleophilic attack of
the active site on the susceptible substrate60.
8
A
B
Figure 1.3. : (A) 3D backbone fold of Fusarium solani pisi cutinase, showing accessibility of the active site
region. The dynamics of the backbone is also indicated. Red, mobile; blue, rigid (from Egmond and Vlieg 61);
(B) Ribbon representation of Fusarium solani pisi cutinase, highlighting two of the four cysteins (Cys-31; Cys109) in the neighborhood of the buried single tryptophan residue (Trp-69) (from Vidinha et al.86).
The aforementioned mobility had already been predicted to allow efficient interaction with
the substrates57, and NMR studies unraveled a micro to millisecond time scale mobility of
the active site60, in which the two loops in a coil-like motion move one plan helix as a whole,
opening and closing access to the substrate binding site. Coincidently, the recorded breathlike movements corresponded to time scale magnitude of hydrolysis reaction kinetics55.
A number of arginine residues at the surface of cutinase are of structural importance as
concerns the behavior of the enzyme in biphasic media. They constitute a positively charged
collar located just beyond the substrate binding region, which keeps cutinase water soluble
while the enzyme exposes a relatively large hydrophobic binding region to the substrate61.
On the other hand, when cutinase adsorbs to a lipid layer, the binding region is immersed in
the lipid phase, while the positively charged collar just remains in the water phase60.
In regard to enzymatic activity, it was observed that cutinase is very sensitive to the position
and chain length of the acyl group in the organic substrates. Kinetics studies with pseudo
tryglicerides containing only one hydrolysable ester bond at position 3 showed that the
activity of cutinase is very sensitive to the length of the chain that is hydrolyzed as well as to
the chain at position 1. The highest activities were found when chains in those positions
contained three or four carbon atoms60;61. The structural explanation for the short chains
preference was provided by crystallography studies, which showed that the chains at such
9
position are completely buried in a rather small pocket: only about five carbon atoms can be
embedded in that pocket62 .
The work developed in this thesis was focused on Fusarium solani pisi cutinase immobilized
in 1:5 TMOS/BTMS (tetramethoxysilane/n-butil-trimethoxysilane) sol-gel matrices. This
comes in line with previous work by Vidinha and co-workers50 who studied the
encapsulation of cutinase in sol-gel matrices of varying composition and found that enzyme
activity was highest for the above precursor combination, lower and higher chain lengths of
the mono-alkylated precursor or decreasing proportions of the latter relative to TMOS
leading to lower enzyme activity. The beneficial effect of BTMS was confirmed in studies
with combinations of three precursors. Vidinha and co-workers50 suggested that the presence
of BTMS in higher proportion than TMOS gave a good compromise between structural
integrity of the material and cutinase/matrix interactions.
Another important parameter already referred as influencing the activity of sol-gel entrapped
enzymes is the amount of water used to hydrolyse the precursors. A quantity is defined to
account for this effect – the water to silane molar ratio (R). Reetz and co-workers53 observed
that at lower R values, the activity of a sol-gel entrapped lipase was low, and the possibility
that enzyme aggregation occurred was put forward. With increasing amounts of water up to
R = 8 to 10, enzyme activity slowly increased, decreasing again at higher R values. The
stoichiometric proportion of water to silane is the crucial requisite to the hydrolysis network
forming step, and the remaining available water will likely influence the catalytic behavior of
the entrapped enzyme molecules.
In this context, the quantity of water available to the enzyme is an important parameter when
a biocatalytic reaction is performed in nonaqueous media. It has been reported that the
addition of a small quantity of water was necessary to insure a proper activation of the
enzyme, in particular to ensure some conformational flexibility65; 66. The rationale is that the
enzymes have rigid structures at low water activity, and become flexible with increasing
water content67. The observed decrease in the catalytic activity of cutinase at higher organic
solvent concentration was attributed to spontaneous denaturation of the enzyme68 by
displacement of bound water molecules by the organic solvent, resulting in a dramatic
change of the protein structure that destroyed the catalytically active enzyme conformation69.
It was already referred herein that encapsulated enzyme molecules must have enough room
to change their conformation as required for the full catalytic cycle. Rotation of sol-gel
10
entrapped enzymes requires that the entrapping cage leave some space between the outer
surface of the protein and the silica surface of the cage. That space should contain also water
molecules, a small reservoir surrounding the protein, much like a blanket-thin layer against
the silica cage wall70 (Figure 1.4).
Figure 1.4. : Schematic view of an entrapped enzyme with a few water molecules inside a sol-gel pore (from
Frenkel-Mullerad and Avnir70).
On the other hand, it is known that low availability of water in the immobilization
microenvironment promotes hydrophobic interactions within polypeptide chains, thus
improving thermal stability of the enzymes71. Consistent with this rationale and apart from
the molecular confinement imposed by adsorption to silica nano-cages, the effect of sol-gel
entrapment on protein stabilization72 has been ascribed to alterations in protein hydration.
The addition of osmolites was referred to affect the folding of the protein by leading to the
disruption of the ordered water structure within silica, and hence enhancing the hydrophobic
effect73. Globally it stands out that the low water content reduces the mobility of enzyme
peptide chains and hinders the unfolding of the enzyme that goes along with denaturation.
On the other hand, low water content can also induce enzyme aggregation. Thus, the right
balance must be found for the availability of water to the sol-gel entrapped enzyme.
11
Vidinha and co-workers74 also correlated the specific activity of sol-gel entrapped cutinase
with the structure of the silica matrices. The matrices were characterized by diffuse
reflectance infrared Fourier transform (DRIFT) spectroscopy and by solid-state
29
Si and 1H
nuclear magnetic resonance (NMR). That study revealed no traces of water in the matrices,
which turned it rather interesting to look more thoroughly at the enzymatic activity and
thermal stability of sol-gel entrapped cutinase. Curiosity was induced as well by knowing the
suggested role of water in preventing molecular aggregation, and bearing in mind that the
catalytic region of cutinase is of hydrophobic character and the enzyme has a positive charge
collar-like arginine sequence by which the enzyme is adapted to biphasic media60;61. Vidinha
and co-workers74 found that for alkylated precursors with chains up to C4 – i.e. up to the
combination 1:5 TMOS/BTMS – there was an increase in the organic content of the sol-gel
matrix (with the consequent decrease in the condensed silica content), accompanied by an
increase also in the residual silanol groups. For TMOS/BTMS matrices, this resulted in high
contents of alkyl (% CHn; ca. 40 %), and silanol (% OH; ca. 25 %), which could
hypothetically mimic the biphasic medium to which cutinase is most adapted. However the
permeability constrains of the immobilized support ought to be seen in a broader sense than
strictly concerning the solvents. While the afore-mentioned CHn content confers lipophylic
character to the matrix, hydroxyl content affects microenvironment polarity74. The relevance
of the last parameter has to do with the fact that an alcohol molecule R-OH can easily adsorb
by hydrogen bonding on the negatively charged surface siloxanes ≡Si-O- or even on neutral
silanols ≡Si-OH, and lose a proton to produce neutral siloxanes ≡Si-OH or protonated
silanols ≡Si-OH2+, in addition to alkoxy anions RO-. These anions would diffuse very fast
towards the enzyme, due to repulsion by negative silica surface charges 44 and accordingly
lead to enhanced alcohol substrate availability to the encapsulated enzymes, likely
influencing its activity.
The present thesis revolves around these issues. It addresses the influence of enzyme loading
on the specific activity of cutinase entrapped in 1:5 TMOS/BTMS matrices, using a model
transesterification reaction in a non-aqueous medium to measure enzyme activity in response
to changes in enzyme loading. To search for evidence of enzyme aggregation, an active site
titration technique was developed, based on the work of Walz and Schwack68. An enzyme
inhibitor was first tested in aqueous media, and the inhibition process was then applied to
sol-gel entrapped cutinase, followed by the assessment of residual enzyme activity. The
12
entrapped enzyme was also submitted to thermal treatment, and the resulting effects on
enzyme specific activity were monitored.
These approaches were complemented by a microscopic characterization of the immobilized
enzyme using fluorescence spectroscopy, which has become a standard technique to follow
conformational modifications of enzymes and monitor enzymatic stability75;76. Fluorescence
spectroscopy has been used extensively to provide information on the dynamics of cutinase
dissolved in aqueous media77;78, adsorbed on solid supports75 or encapsulated in reverse
micelles78; 79; 80. Fluorescence emission may have contributions from three types of aromatic
residues – tyrosine, tryptophan and phenylalanine – but typically the phenylalanine quantum
yields are too low to be detected81;82. In the case of cutinase, which possesses six tyrosine
residues dispersed around the active site and one tryptophan residue located in the opposite
region (Figure 1.3), the intrinsic fluorescence is dominated by the tyrosine residues 48 rather
than by the tryptophan. That feature has been attributed to the disulfide bond located only 4
Å away from the tryptophan residue (Trp-69) that is likely to quench the respective
fluorescence60;83. When cutinase is denatured, Trp-69 is removed from the quenching effect
of the disulfide bond between cysteine Cys-31 and Cys-109, becoming exposed to the
solvent77. Thus following the fluorescence spectrum of immobilized cutinase at
progressively higher temperatures may provide insight into conformational changes, as
evidenced by increased emission of free cutinase48, whereby through the selective excitation
of the single tryptophan residue the fluorescence emission becomes dominated by the
tryptophyl77;85.
Since fluorescence anisotropy can provide information on molecular size and shape, it was
thought to be an ideal technique for examining the behavior of sol-gel entrapped cutinase. In
this thesis, fluorescence anisotropy decays were measured at different enzyme loadings, and
fitted by a sum of a one exponential decay plus a constant that considers the residual
anisotropy. The order parameter, S2, can provide information on the degree of mobility of the
fluorescence probe, Trp-69. S2 was measured as a function of enzyme loading, in search of a
correlation consistent with a higher degree of packing of enzyme molecules, or enzyme
aggregation, for more heavily loaded sol-gel matrices.
Tyrosine residues have a low sensitivity to changes in local polarity conditions. On the other
hand, tryptophan residues are very suitable probes to assess the conformational state of
enzymes, since their emission peak is highly dependent on the polarity of their
13
surroundings83;84. Vidinha et al.86 took advantage of that fact to study sol-gel matrices with
the help of steady-state fluorescence spectroscopy. They used Trp-69 as a probe to assess the
polarity of 1:5 TMOS/n-alkyl-TMS matrices already characterized in terms of enzyme
activity. The emission from tryptophan residues shifts to lower wavelengths (blue shift) in
hydrophobic microenvironments87. The sequential decrease in the fluorescence emission
intensity maximum (λmax) recorded for sol-gel matrices up to TMOS/BTMS was indicative
of increased hydrophobicity. Vidinha et al.86 also looked at the permeability of the sol-gel
matrices, namely at the quickness of response of Trp-69 to the immersion of the sol-gel
matrix in a given medium, and corresponding shift in λmax. They saw that Trp-69 responded
to microenvironment polarity changes induced by methanol, acetonitrile and n-hexane, but
not water alone.
In this thesis, steady-state fluorescence spectroscopy was used to look for evidence of
cutinase denaturation upon thermal treatment of the sol-gel matrices with entrapped cutinase.
When cutinase is in aqueous solution, it unfolds as temperature increases, with concomitant
increases in fluorescence emission87. On the other hand, time resolved fluorescence
spectroscopy allowed the determination of the decay times of dissolved cutinase87. These
increased with increasing temperature, as denaturation took place. Thus in this thesis, time
resolved fluorescence spectroscopy was used as well, to study the impact of temperature on
the enzyme via conformational changes, in matrices with varying degrees of enzyme
loading.
In this thesis, the microscopic characterization of the sol-gel matrices used for entrapping
cutinase was also carried out using NMR spectroscopy. As noted above, Vidinha et al.86 saw
that Trp-69 was sensitive to the permeation of sol-gel matrices by some solvents, but not all.
These findings led us to carry out a study of diffusion within the sol-gel matrices, using High
Resolution Magic Angle Spinning (HR-MAS) Pulsed Field Gradient (PFG) NMR
spectroscopy. The technique, which very few laboratories in the world have available, is
based on the principle that each proton spin in a magnetic field is characterized by a specific
frequency determined by the magnetic field strength. If a magnetic field gradient is applied
with spatial-dependent field intensity, then the frequency effect of each proton spin will also
be position-dependent. This allows the determination of the diffusion coefficients of the
different species in the system. In solid samples, and to avoid line-broadening effects on
NMR spectra, samples have to be spun at high rate, at the magic angle (54.7°). The aim of
this study was to measure the permeation of the compounds of interest in the solvent used in
14
the specific enzymatic activity assays50 – n-hexane – as well as the solvent used in the active
site titration protocol – acetonitrile. A more polar solvent – methanol – was also selected for
comparison. Additionally, the influence of the presence of enzyme in the sol-gel matrix was
looked at.
1.3. Structure of the thesis
The issues discussed in this thesis are organized in three core sections. The first one,
corresponding to Chapter 2, is entitled “Assessing the aggregation of cutinase in sol-gel
matrices”. Cutinase activity was monitored with a model transesterification reaction used in
previous studies published by our laboratory50;74;86, to allow comparison. Once the
previously mentioned trend of decreasing specific enzyme activity with increasing protein
concentration64 was confirmed, the hypothesized aggregation phenomenon of the
immobilized enzyme molecules was investigated by performing irreversible inhibition
studies. The results obtained showed that the number of catalytically active sites of the
immobilized enzyme decreased as enzyme loading in the matrices increased. Fluorescence
anisotropy decay measurements confirmed a higher degree of packing of the enzyme
molecules in the more heavily loaded sol-gel matrices, consistent with enzyme aggregation,
while HR-MAS PFGSE NMR spectroscopy revealed that the presence of the enzyme did not
affect the diffusion of solvents within the sol-gel matrices.
The second core section, corresponding to Chapter 3, is entitled “Thermal stability of sol-gel
entrapped cutinase”. It focuses on the response of the entrapped enzyme to thermal
treatment, including incubating sol-gel matrices with varying levels of enzyme loading at
discrete temperatures up to 100 ºC, for 24 hours. Enzymatic assays were performed to assess
the impact of thermal treatment on cutinase activity, and steady-state fluorescence
spectroscopy was used to assess any signs of enzyme denaturation inside the matrices via
extensive conformational changes. Time resolved fluorescence spectroscopy confirmed that
enzyme denaturation, as known to occur at identical temperatures for cutinase in solution,
did not take place when cutinase is entrapped in sol-gel matrices. The latter do have a
protective effect on cutinase, but further studies will be needed to clarify that effect.
15
The third core section, corresponding to Chapter 4, is entitled “Solvent mobility in sol-gel
matrices as measured by HR-MAS PFGSE NMR spectroscopy”. It reports on the
measurement of diffusion coefficients for solvents of different polarities, as well as a
substrate species and one other species that although not being substrate nor product, can
have a very strong impact on the catalytic activity of sol-gel entrapped enzymes and enzyme
action in non-aqueous media – water. Assays were performed with liquid samples: the
solvents alone, or the solvents with substrate or with water. Experiments were also
performed with sol-gel matrices embedded with the liquid samples. The results for the liquid
samples revealed the expected mono-exponential decays of the echo-spin signals of
solutions. There were also bi-exponential decays registered with some samples in the
presence of the matrix, which may evidence the existence of two limiting diffusional
domains. Such phenomena have occurred with those species whose diffusion was most
affected by the chemical interaction with the matrix milieu.
16
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52 - Gill, I., Ballesteros, A. 2000. Bioencapsulation within synthetic polymers (Part 1):
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entrapment in hydrophobic sol-gel materials. Biotechnol. Bioeng. 49, 527 – 534.
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Solvent . Nature. 356; 615-618.
58 - Martinez, C., Nicolas, A., van Tilbeurgh, H., Egloff, M.,Cudry, C., Cambillau, C. 1994.
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59 - Van Tilbeurgh, H., Egloff, M., Martinez, CF., Rugani, N., Verger, R., Cambilau, C.
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H., Hilbers, C., Pepermans, H. 1999. NMR Studies of Fusarium solani pisi Cutinase in
Complex with Phosphonate Inhibitor. Biochemistry, 38, 5982-5994.
61 - Egmond, M., Vlieg, J. 2000. Fusarium solani pisi cutinase. Biochimie 82, 1015-1021.
62 - Longhi, S., Czjzek, M., Lamzin, V., Nicolas, A., Cambillau, C., 1997. Atomic
resolution (1.0 angstrom) crystal structure of Fusarium solani cutinase: Stereochemical
analysis. J. Mol. Biol. 268, 779-799.
63 – Reetz, M., Zonta, A., Simpelkamp, J., Rufinska, A., Tesche, B. 1995. Characterization
of Hydrophobic Sol-Gel Materials Containing Entrapped Lipases. L. Sol-Gel Sc. Tecnh.
7: 35 – 43.
64 - Reetz, M., Zonta, A, Simpelkamp, J. 1995. Efficient heterogeneous biocatalysts by
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65 - Pencreac’h, G., Baratti, L. 1997. Activity of Pseudomonas cepacia lipase in organic
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66 - Pencreac’h, G., Baratti, L. 1999. Properties of free and immobilized lipase
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67 - Furukawa, S., Ono, T., Ijima, H., Kawakami, K. 2001. Enhancement of Activity of
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68- Walz, I., Schwack W. 2006. Cutinase Inhibition by means of insecticidal
organophosphates and carbamates; Part 1: Basics in development of a new enzyme
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69 - Mozhaev, V. 1999. Engineering stability of enzymes in systems with organic
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of biocatalysts. Elsevier Science, Amsterdam, pp 355-363.
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71 - Monsan, P., Combes, D. 1988. Enzyme stabilization by immobilization. Methods in
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72 – Eggers, D., Valentine, J. 2001. Crowding and hydration effects on protein
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Acid Additives to Stabilize Enzymes within Sol-Gel Derived Silica. Chem. Mater. 15: 737
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74 – Vidinha, P., Barreiros, S., Cabral, J., Nunes, T., Fidalgo, A., Ilharco, L. 2008.
Enhanced Biocatalytic Activity of ORMOSIL-Encapsulated Cutinase: The Matrix
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Poly(methylmetacrylate) Latex Particles. J. Biotecnhol. 102: 241 – 249.
76 - Guo, Q., Zhao, F. Guo S-Y., Wang X. 2004. The Tryptophane Residues of Dimeric
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77 - Melo, E., Faria, T., Martins, L., Gonçalves, A., Cabral., J. 2001. Cutinase Unfolding
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78 – Ternstörm, T., Svendsen, A., Akke, M., Adlercreutz, P. 2005. Unfolding and
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79 – Gonçalves, A., Aires-Barros, M., Cabral, j. 2003. Interaction of an anionic surfactant
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80 - Melo, E., Baptista, R., Cabral., J. 2003. Improving Cutinase Stability in Aqueous
Solution and in Reverse Micelles by Media Engineering. J. Mol. Catal. B: Enzim. 22: 299
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3585.
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23
Chapter 2
Assessing the aggregation of cutinase in solgel matrices*
2.1. Abstract
Cutinase from Fusarium solani pisi was entrapped in sol-gel matrices of composition 1:5
tetramethoxysilane:n-butyltrimethoxysilane (TMOS/BTMS), and its specific activity was
measured as a function of enzyme loading in the range of ca. 0.1 % to 7 %. To elucidate the
pronounced increase in specific activity that was observed as enzyme loading decreased, an
active site titration technique was applied, based on enzyme inactivation by the inhibitor
paraoxon. It was found that the number of available enzyme activate sites decreased as
enzyme loading in the matrices increased, suggesting that enzyme aggregation occurred in
the more heavily loaded sol-gel supports. The impact of enzyme loading on the packing of
cutinase inside the matrices was studied by fluorescence spectroscopy based on the single
tryptophan residue of cutinase. Fluorescence anisotropy decay measurements led to
relatively low order parameters, indicating that the restrictions imposed on tryptophan by its
microenvironment are small, which is consistent with the fact that tryptophan is only
partially exposed on the cutinase molecule. The order parameter increased as enzyme
loading increased, indicating loss of mobility of the tryptophan in the more heavily loaded
sol-gel matrices, which is consistent with enzyme aggregation. The effect of enzyme loading
on solvent mobility within the matrix was assessed by determining the self-diffusion
coefficient of the solvent using Pulsed Field Gradient Spin Echo High Resolution Magic
Angle Spinning NMR spectroscopy (PFGSE HR-MAS NMR). The diffusion coefficients
determined indicate that enzyme loading has a negligible effect on solvent mobility.
__________________________________________________________________________________
* This chapter has been submitted for publication. Co-authors and affiliations: Gustavo Barreira1, Pedro
Vidinha1, Eurico J. Cabrita1, Joaquim M.S. Cabral2, José M.G. Martinho3, João Carlos Lima1, Susana
Barreiros1; 1REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; 2IBB-Instituto de Biotecnologia e Bioengenharia,
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, 1049-001, Lisboa, Portugal; 3Centro
de Química-Física Molecular, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
24
2.2. Introduction
Enzymes are very versatile catalysts that effectively and precisely regulate processes in
living organisms1. Enzymes often display high chemo-, regio- and stereo-selectivity under
mild reaction conditions2,3, which can be exploited advantageously in many commercial
applications. However, this requires sufficient enzyme stability. Improvements in the latter
property can greatly impact on the economic viability of enzymatic processes. The
immobilization of enzymes in porous matrices can protect the catalysts from aggressive
medium conditions4 and prevent the leaching of the enzyme, while ensuring adequate access
of substrates and release of products to and from the enzyme. The sol-gel process has been
extensively used as a technique for entrapment of enzymes, for use in such diverse fields as
biomedical applications5, lactose intolerance treatments6, artificial organs design7, antibiotics
production8,9, biomass hydrolysis10, bioremediation11-13, chemical sensors and biosensors1420
.
Sol-gels are highly porous materials formed through the hydrolysis and condensation of
metal alkoxides23. The encapsulation of enzymes in silica sol-gels prepared by hydrolytic
polymerization of tetraethoxysilane was pioneered by Avnir and co-workers24, and has been
used ever since in the immobilization of a huge variety of biomolecules25. As the enzymes
are added at the early stage of silicate formation, the sol-gel approach results in a fairly
stable form of enzyme immobilization, biocatalysts being trapped within a silica cage
tailored to their size and shape4. The mobility of the enzyme molecules confined within the
sol-gel pores is thus restricted, and can prevent the unfolding and denaturation of the
encapsulated enzyme26. Notwithstanding such stability improvements, the immobilization of
an enzyme on/in a carrier often leads to a loss of more than 50% activity27, especially at high
enzyme loadings28, which beyond a certain critical amount do not result in higher catalytic
activity29. Nonetheless, sol-gel entrapment has led to remarkable improvements in enzyme
performance in organic solvents30, as evidenced by the encapsulation of lipases31 or a
structurally related lyase32 in which rate enhancements of 2-8 fold were registered, compared
with the free enzymes.
25
The morphologies of the sol-gel matrices depend on the method of drying33. Drying by
evaporation affords the so-called xerogels, in which capillary stress causes shrinkage of the
nano-cages and pores. However, when alkylsiloxanes RSi(OR)3 are used together with
Si(OR)4, the surface of the sol-gel is more densely populated by the hydrophobic alkyl
groups, and the capillary stresses that operate on the entrapped enzymes during evaporation
are largely attenuated34. In this line of work, we previously studied the effect of sol-gel
precursors on the catalytic activity of the enzyme cutinase, immobilized in sol-gel matrices
prepared from different combinations of Si(OMe)4 (TMOS) and n-alkyl-Si(OMe)3 (n-alkylTMS: M-methyl; P-propyl; B-butyl; OC-octyl), and showed that enzymatic activity was
highest in 1:5 TMOS/BTMS sol-gel matrices35.
Cutinase is produced by the phytopathogenic fungus Fusarium solani pisi, and is one of the
smallest members of the serine hydrolase family36. Pathogenic fungi produce and secret the
enzyme to hydrolyse cutin, an insoluble lipid polyester matrix that covers plant surfaces37.
Cutinase is a protein with 197 amino acid residues, whose crystal structure has been solved
to high atomic resolution (1 Å). Cutinase is an α/β hydrolase with a core formed by five
parallel -strands surrounded by four α-helices. The catalytic site has three residues –
Ser120, Asp175 and His188 – and an oxyanion hole36. Recently inhibition studies of
Fusarium solani pisi cutinase with diethyl-p-nitrophenyl phosphate (paraoxon) have been
reported38, and the inhibitory potential revealed by paraoxon was correlated with the electron
deficiency at the central phosphorus of the inhibitor molecule as a result of the leaving group
chemistry. The polarization of the P=0 bond facilitates the attack of nucleophilic agents, such
as the hydroxyl group of Ser120, forming a covalently bonded phosphorylated enzyme, as
evidenced by the three-dimensional structure of cutinase complexed with phosphonate
inhibitors39. The high specificity of paraoxon towards the active site serine makes this
inhibitor an attractive reagent for active-site titration.
Following studies in which we used several techniques to correlate the specific activity of
sol-gel encapsulated cutinase with its microenvironment40; 41 we now focus on the relation
between enzyme loading in the sol-gel supports and cutinase specific activity. We first
measured that relationship, and then used several techniques to account for the experimental
results: active-site titration of the sol-gel entrapped enzyme; fluorescence anisotropy
measurements, based on the single Trp residue of cutinase, to look for microscopic evidence
of changes in the packing of cutinase molecules with changes in enzyme loading; Pulsed
Field Gradient Spin Echo High Resolution Magic Angle Spinning NMR spectroscopy
26
(PFGSE HR-MAS NMR), to assess solvent diffusion in enzyme loaded sol-gel matrices vs.
sol-gel matrices without enzyme 42; 43.
2.3. Materials and methods
2.3.1. Materials
Fusarium solani pisi cutinase was produced by an Escherichia coli WK-6 (a gift from
Corvas International, Ghent, Belgium) and purified at Centro de Engenharia Biológica e
Química, Instituto Superior Técnico44;45. The enzyme purity was controlled by
electrophoresis and isoelectric focusing. (R,S)-2-phenyl-1-propanol (97 % purity), (R,S)-2phenyl-1-propyl butyrate (98 % purity), tetramethoxysilane (TMOS) and paraoxon (diethyl
4-nitrophenyl phosphate; 90 % purity) were from Aldrich, n-butyltrimetoxysilane (BTMS)
was from Polysciences Inc., vinyl butyrate (99 % purity) was from Fluka, n-hexane,
tridecane and acetonitrile (99.9 % purity) were from Merck, Hydranal Coulomat A and C
Karl-Fischer reagents and Tris (99.5 % purity) were from Riedel de Häen, polyvinyl alcohol
(PVA; MW 15.000) and p-nitrophenyl butyrate (pNPB; 98 % purity) were from Sigma, HCl
(37 % purity) was from Panreac, methanol (99.8 % purity) was from Carlo Erba.
2.3.2. Cutinase immobilization in sol-gel
Following Vidinha and co-workers35 a typical procedure consisted in preparing separately, in
eppendorfs, an aqueous solution containing the enzyme (a given amount of lyophilized
cutinase added to 58 L of 1 M NaF solution, 116 L of 4 % w/v PVA solution and 265L
of distilled-deionised water, making a total of 24.36 mmol of water) and a mixture of
precursors (76 L of TMOS and 487L of BTMS, to yield a TMOS:BTMS molar ratio of
1:5 and a water/silane molar ratio of 8), adding the latter to the former, under vigorous
shaking on a vortex mixer, until the mixture became homogeneous. The content of the
eppendorf was poured rapidly onto a parafilm-covered plastic tube. The tube was placed in
an ice bath, and kept there for 10 minutes while gelation took place, after which it was kept
at 4 ºC for 24 h. The parafilm holding the material was then placed on a glass dish for air
drying at 35 ºC for 24 h. The resulting xerogel was fragmented and put in an eppendorf that
27
was placed inside a high pressure cell and submitted to an atmosphere of supercritical CO 2 at
40 ºC and 100 bar, for 6 h. The resulting material was left to age at ambient conditions for 5
days before being ground in a mortar, assayed for the amount of immobilized protein, as
determined by a modified Lowry method58 involving pre-treatment with concentrated NaOH
solution at high temperature to release the protein from the matrix, and used for reaction. To
prepare sol-gel matrices with lower enzyme loadings, the above procedure was slightly
modified: lyophilized enzyme was replaced by the required amount of enzyme dissolved in
264 L of water. No loss of xerogel material was associated to events other than the removal
of fluids upon drying. The reproducibility of the above procedures for preparing sol-gel
matrices was confirmed by the good agreement between measurements of the amount of
immobilized protein, irrespective of enzyme loading. It was verified that keeping samples at
ambient conditions for up to a month did not impact significantly on enzyme activity.
2.3.3. Enzyme activity assays in transesterification reactions
Reactions were performed in n-hexane, in glass vials (reaction volume of 2 mL) placed in a
constant temperature (22 ºC) orbital shaker set for 400 rpm. The reaction studied was the
transesterification of vinyl butyrate (300 mM) by (R,S)-2-phenyl-1-propanol (100 mM). The
addition of the ester marked the start of reaction. The substrates and the solvent were dried
with molecular sieves. Tridecane (20 mM) was used as external standard for GC analysis.
2.3.4. Reutilization assays
For reutilization assays, the liquid medium was pipetted out at the end of reaction and fresh
n-hexane was added to the sol-gel powder. After 24 h at room temperature, the solvent was
removed and the sol-gel powder was dried for 24 h in an oven kept at 40 ºC, before being
weighed and assayed again for enzyme activity. Before adopting this procedure, it was
verified that keeping sol-gel supports immersed in n-hexane for up to 2-3 days at room
temperature did not impact on enzyme activity. Supports were assayed in triplicate for their
enzyme loading, every 2-3 reutilizations.
28
2.3.5. Enzyme particle sizes
Enzyme particle sizes were determined for three different enzyme loadings and found not to
depend on the latter parameter, averaging 12040m. At the 18th reutilization cycle, this
value had dropped to 80 µm ± 20 µm. Smaller particles (powders) were discarded and were
not used for subsequent assays or for measurements of the amount of immobilized protein.
2.3.6. Transesterification reaction analysis
The reaction conversion was measured by GC analysis performed with a Trace 2000 Series
Unicam gas chromatograph equipped with a 30m x 0,32 mm i.d. fused silica capillary
column coated with a 0.25 µm thickness film of 20% 2,3-dimethyl-6-tert-butyldimethylsilylβ-cyclodextrin dissolved in BGB-15, from Analytik AG. Oven temperature program: 60-180
ºC ramp at 4 ºC min-1, 180-220 ºC ramp at 10 ºC min-1, and holding at 220 ºC for 5 min.
Injection temperature: 250 ºC. Flame ionization detection (FID) temperature: 250 ºC. Carrier
gas: helium (2.0 cm3 min-1). Split ratio: 1:20. No products were detected in assays carried out
without enzyme. The initial rates given (per mg of protein) are the average of at least two
measurements.
2.3.7. Free enzyme inhibition assays
We used a protocol similar to that presented by Walz and Schwack38. We prepared a 4.23E05 M solution of cutinase in 500 mM Tris/HCl buffer at pH 7.0 and a stock solution of
paraoxon in methanol that was used to prepare more diluted solutions with concentrations of
paraoxon ranging from 3.63E-03 to 1.82E-04 M. Dilution was performed with de-ionized
water. The concentration of methanol was kept at 10 % (v/v) in all the paraoxon solutions. A
60 mM solution of pNPB in acetonitrile was also prepared. In one set of experiments aimed
at determining cutinase residual activity after inhibition by paraoxon, to a 2 mL polystyrene
cuvette were added 650 µL of Tris/HCl buffer at pH 7.0, 250 µL of inhibitor solution and
100 µL of cutinase solution. The mixture was incubated in the absence of light at room
temperature for 30 min, after which 10 µL of pNPB solution were added. Absorbance was
measured at λ = 405 nm. Two blanks were performed: one in which the cutinase solution
was replaced by Tris/HCl buffer, for determining the extent of non-enzymatic paraoxon
29
hydrolysis, and another in which the inhibitor solution was replaced by the buffer, for
determining maximum cutinase activity. Assays were done in triplicate. The extinction
coefficient of paraoxon was taken as 12351 M-1 cm-1 (38).
2.3.8. Immobilized enzyme inhibition assays
We prepared a 1.0E-03 M stock solution of paraoxon in acetonitrile that had been dried with
molecular sieves. Sol-gel matrices without enzyme were incubated in 1 mL of paraoxon
solution in 2 mL polystyrene cuvettes, in the absence of light and at room temperature, for up
to 10 h. Absorbance was measured at λ = 405 nm, at regular time intervals. In experiments
aimed at determining cutinase residual activity after inhibition by paraoxon, to a 5 mL glass
vial were added ca. 20 mg of sol-gel preparations with a given enzyme loading, the amount
of paraoxon stock solution required to set a 1:10 enzyme:inhibitor molar ratio, and
acetonitrile to a final incubation volume of 2 mL.The mixture was incubated in the absence
of light at room temperature overnight, after which the liquid medium was pipetted out and
n-hexane was added to the sol-gel powder. After 24 h at room temperature, the solvent was
removed and the sol-gel powder was allowed to dry for an extra 24 h, before being weighed
and assayed for enzyme activity in transesterification reactions in n-hexane. This procedure
was applied to sol-gel matrices with varying enzyme loadings. A set of blanks for
determining maximum cutinase activity was obtained by submitting sol-gel matrices to the
same series of experimental steps while replacing the inhibitor solution with acetonitrile,
followed by testing in transesterification reactions in n-hexane. Two of the immobilized
enzyme preparations were also assayed in the hydrolysis of pNPB in acetonitrile, as
described in free enzyme inhibition assays. Acetonitrile was used with ca. 1 % (w/w) water
concentrations (water activity of ca. 0.2), as measured by Karl-Fischer titration.
2.3.9. Fluorescence anisotropy decays
Time-resolved pico-second fluorescence measurements were performed using the singlephoton counting timing method with laser excitation. The setup consisted of a mode-locked
Spectra-Physics Vanguard 2000-HM532 Nd:YVO4 diode laser, delivering 2 Wof 533 nm
light at a repetition rate of 76 MHz and pulse duration of ≈12 ps that synchronously pumped
a cavity dumped 710-2 dye (rhodamine 6G) laser, delivering 3-4 ps pulses at a repetition rate
of 1.9 MHz. The laser light was frequency doubled using a LBO crystal to obtain laser light
30
of 280 nm used for excitation. Intensity decay measurements were made by an alternate
collection of impulse and decay, with the emission polarizer set at the magic angle position.
Impulse was recorded slightly away from the excitation wavelength with a scattering
suspension. For the decays a cutoff filter was used, effectively removing all excitation light.
The polarized decays were measured by alternate collecting of the horizontal and vertical
components in successive cycles of 30 s until the number of counts at the maximum of the
vertical component reached 20.000 counts. The global time of acquisition for each
component was rigorously equal. The time per channel was 19.5 ps and the number of
channels used in the multichannel analyzer was 1024. The emission signal was first passed
through a depolarizer, and then sent to a Jobin-Yvon HR320 monochromator with a grating
of 100 lines/nm, and was recorded on a Hamamatsu 2809U-01 micro-channel plate
photomultiplier as a detector. The instrument response function had an effective fwhm of 35
ps. The numerical aperture for fluorescence collection was 0.18 (lens with a diameter of 18
mm and a focal length of 50 mm); hence the half-angle θ was 10.4°, and paraxial conditions
hold. The effect of a finite collection cone on the measured anisotropy was negligible46 .The
anisotropy decay curves were constructed from the IVV (t), IVH(t) fluorescence decays
obtained with vertical polarized excitation light and selecting the vertical (IVV(t)) or
horizontal (IVH(t) ) components of the fluorescence.
r (t ) 
I VV (t )  GIVH (t )
I VV (t )  2GIVH (t )
(1)
Where G=IHV(t)/IHH(t) is an experimental correction factor that considers the artifacts
introduced by the detecting system on the polarized fluorescence light components. For our
experimental setup G=1, because the polarized fluorescence light was depolarized before the
entrance slit of the monochromator.
31
2.3.10. HR-MAS NMR diffusion spectroscopy
All NMR experiments were performed using a Bruker Avance III 400 operating at 400.15
MHz for protons, equipped with a 4 mm high-resolution solid-state Magic Angle Spinning
(MAS) probe and with pulse gradient units, capable of producing magnetic field pulsed
gradients in the z-direction of 0.54 T.m-1. Samples were spun at the magic angle at a rate of 4
kHz, the experimental temperature determined under these conditions was 23 ºC and was
constant within ± 0.1 ºC, as measured using the spectrometer thermocouple system. The
spectra were recorded in 12L capacity 4 mm ZrO2 rotors. Diffusion measurements were
performed using the stimulated echo sequence using bipolar sine gradient pulses and eddy
current delay before the detection 47. The signal attenuation is given by
æ
æ d ææ
I = I 0 exp æ-g 2 g2d 2 D æD - ææ
æ 3 ææ
æ
(2)
where D denotes the self-diffusion coefficient,  the gyromagnetic ratio,  the gradient pulse
width,  the diffusion time and g the gradient strength corrected according to the shape of
the gradient pulse.Typically, in each experiment a number of 32 spectra of 32K data points
were collected, with values for the duration of the magnetic field pulse gradients (δ) of 1.0 to
2.0 ms, diffusion times (Δ) of 50 to 150 ms and an eddy current delay set to 5 ms, the
gradient recovery time was 200 s. The sine shaped pulsed gradient (g) was incremented
from 5 to 95% of the maximum gradient strength in a linear ramp. The spectra were first
processed in the F2 dimension by standard Fourier transform and baseline correction with
the Bruker Topspin software package (version 2.1). The diffusion coefficients are calculated
by exponential fitting of the data belonging to individual columns of the 2D matrix using
Origin 1.2 data software program. Whenever possible the diffusion coefficients (D) were
obtained by measuring the signal intensity at more than one resonance in the spectra and the
average value was used. At least two different measurements were done for the
determination of each diffusion coefficient.
32
2.4. Results and Discussion
In an earlier paper40, we used TMOS/BTMS supports with an average enzyme loading of 1.4
%, which was similar to the values obtained for adsorption of cutinase on zeolite NaA, and
slightly lower than for adsorption of cutinase on zeolite NaY. As we commented at the time,
doubling the enzyme loading in the sol-gel supports did not have a marked effect on the
specific activity of the enzyme. However, we did not look at the effect of lower enzyme
loadings on the catalytic behaviour of cutinase. Figure 2.1. shows results obtained for sol-gel
supports with cutinase loadings varying from 0.08 % to 3.7 %. We readdressed the issue of
enzyme stability by submitting the sol-gel supports with different enzyme loadings to a
number of successive assays. Figure 2.1. shows the results obtained for supports freshly
prepared, as well as the same supports in the 2nd to 6thutilization.
initial rate/nmol min-1 mg-1
300000
250000
200000
150000
100000
50000
0
0
1
2
3
4
enzyme loading/% (w/w)
Figure 2.1. : Impact of enzyme loading of sol-gel matrix on cutinase specific activity. Five matrices with
enzyme loadings of 0.08 %, 0.27 %, 0.52 %, 0.97 % and 3.65 % were prepared and used six times
consecutively in a transesterification reaction performed in n-hexane at room temperature, yielding the six data
sets shown in the figure., 1st; , 2nd; ▲, 3rd; △, 4th; ●, 5th; ○, 6th. The lines are trend-lines. The standard
deviations of enzyme loading values range from ca. 17 % for higher enzyme loadings, to ca. 32 % for the two
lowest enzyme loadings.
As the figure shows and as expected, enzyme activity remained fairly constant during the
reutilization cycle period. The impact of enzyme loading on the specific activity of the
enzyme was very modest for enzyme loadings above 1.5 %, but was very pronounced for
33
lower values of the latter parameter. It is remarkable how a decrease in enzyme loading
below the range of 1-3 %, so commonly found in immobilized enzyme preparations, can
have such a pronounced effect on the catalytic response of a sol-gel entrapped enzyme.
Whatever the immobilization technique, it is expected that beyond a certain enzyme/support
ratio there will be no further gains in the specific activity of the enzyme. In their early
studies on sol-gel entrapped enzymes, Reetz and co-workers29 who observed a similar
behaviour to that depicted in Figure 2.1, rationalized the decrease in specific activity
observed for higher enzyme loadings on the basis of diffusional limitations of the substrates,
as well as the aggregation of enzyme molecules, and hence a lower degree of dispersion of
the catalyst.
One of the effects of the aggregation of enzyme molecules at higher enzyme loadings can be
a decrease in the number of available catalytic sites. To search for a possible correlation
between the latter and the enzyme loading in the sol-gel matrices, we followed the protocol
developed by Walz and Schwack38. Their strategy is based on the use of a species that causes
irreversible inhibition of cutinase, and measuring residual enzyme activity after a given
number of enzyme molecules has been exposed to varying concentrations of the inhibitor.
We used one of the inhibitors referred by the authors, namely paraoxon, an
organophosphorous insecticide. One molecule of paraoxon binds irreversibly to the hydroxyl
of the Ser120 residue of the active site triad48;49 forming a covalently bonded phosphorylated
enzyme38.We first used this protocol to perform active site titration for cutinase dissolved in
aqueous buffer. As remarked by Walz and Schwack38, although cutinase exhibits highest
activity at pH 8.5(50) it is still sufficiently reactive at neutral to moderately alkaline pH, at
conditions where organophosphorous insecticides are still stable enough to conduct the
inhibition assays 51. On the other hand, the enzymatic assays following enzyme inhibition are
based on the hydrolysis of pNPB, and nitrophenyl esters are stable only in the range of acidic
to neutral pH52.
The binding of the phosphorus center of paraoxon to the enzyme active site leads to the
release of one molecule of p-nitrophenol. Due to the range of inhibitor concentrations used,
the inhibition process itself was not expected to interfere with the subsequent enzyme
activity assays. In fact, the increase in the concentration of p-nitrophenol in the blank assay
without enzyme was proportional to the increase in inhibitor concentration, but was always
low, with absorbance values not exceeding 0.02. Although all manipulations involving
paraoxon were performed at conditions that minimized exposure to light, the high
34
photosensitivity of paraoxon led to a small level of degradation of the inhibitor in all the
experiments. After incubation of the enzyme solutions with the inhibitor, the residual activity
of the enzyme was determined by adding pNPB and measuring the release of p-nitrophenol.
The second blank, without inhibitor, was used to set 100 % residual enzyme activity.
Figure 2.2. : Residual activity of free cutinase in aqueous buffer after exposure to varying concentrations of the
inhibitor paraoxon. Enzyme activity was measured by adding pNPB and measuring the release of p-nitrophenol
at λ = 405 nm. [cutinase] = 4.2E-06 M. The line is a trend-line.
As seen in Figure 2.2, an enzyme:inhibitor molar ratio of 1:10 caused ca. 30 % inhibition of
enzyme activity in the case of free cutinase. The number of available active sites of cutinase
should be maximal in aqueous buffer. If the number of catalytically competent sites of solgel entrapped cutinase is indeed higher for lower enzyme loadings, then by using a 1:10
enzyme:inhibitor molar ratio, enzyme inhibition should be more severe in the matrices more
heavily loaded with cutinase, and should not exceed 30 % in the less heavily loaded
matrices. The inhibition of sol-gel entrapped enzyme could not be performed using nhexane, the solvent used in the model enzymatic transesterification reactions. The procedure
requires a solvent that is hydrophilic enough to dissolve paraoxon, but not so hydrophilic that
it will not permeate the relatively hydrophobic sol-gel matrix40. On the other hand, exposure
of the enzyme to the chosen solvent must not impact too severely on enzyme activity.
Acetonitrile meets these criteria. We have evidence that it easily permeates the sol-gel
matrices used in the present study through measurements of the fluorescence emission
35
intensity maximum (max) of the single tryptophan residue of cutinase41. To assess the effect
of acetonitrile on the entrapped enzyme, we incubated immobilized enzyme samples in
acetonitrile for 24 h, after which the samples were washed with n-hexane and tested for
catalytic activity, revealing that exposure to acetonitrile did not have a negative impact on
cutinase activity. We also assayed the activity of sol-gel entrapped cutinase in the hydrolysis
of pNPB in this solvent (Table 2.1). Reaction rates followed the same trend of Figure 2.1, i.e.
lower enzyme loadings promoted higher specific rates. The size and polarity of the paraoxon
molecule are not too dissimilar to those of pNPB, and thus the inhibitor should be able to
access the enzyme inside the matrix.
Table 2.1. : Impact of enzyme loading of sol-gel matrix on cutinase specific activity. The two matrices were
assayed in acetonitrile, in the hydrolysis of pNPB at room temperature. Standard deviations of enzyme loading
values as in Figure 2.1.
Reaction rate/nmol min-1 mg-1
Enzyme loading/%
16.0
0.17
71.9
0.06
As indicated earlier, a 1:10 enzyme:inhibitor molar ratio was selected for the inhibition
assays of sol-gel entrapped cutinase. Incubation of the sol-gel matrix without enzyme in
solutions of paraoxon in acetonitrile resulted in levels of paraoxon hydrolysis that were too
low to affect the measurements with enzyme loaded matrices. As with the free enzyme,
assays were performed in the absence of inhibitor, to set 100 % residual enzyme activity.
Our results (Figure 2.3.) show that the inhibitory effect of paraoxon was inversely
proportional to the enzyme loading in the sol-gel matrices. In the matrices carrying less
enzyme, the level of inhibition was very similar to that achieved with the free enzyme (ca. 38
% inhibition), indicating similar availability of catalytic sites. In the case of the matrix with
6.8 % enzyme loading, the same enzyme:inhibitor molar ratio of 1:10 caused ca. 89 %
inhibition of enzyme activity, indicating that the number of catalytically competent sites was
overestimated; the inhibition of 38 % of enzyme active sites would have required an
enzyme:inhibitor molar ratio lower than 1:10. This suggests that cutinase aggregation does
occur in the sol-gel matrices, causing a significant loss of catalytic performance for typical
working ranges of 1-3 % enzyme loading.
36
Figure 2.3. : Residual specific activity of sol-gel entrapped cutinase after exposure, in acetonitrile, to
concentrations of the inhibitor paraoxon setting a 1:10 enzyme:inhibitor molar ratio. Enzyme activity was
measured in a transesterification reaction performed in n-hexane at room temperature. Five matrices with
enzyme loadings of 0.05 %, 0.10 %, 0.47 %, 1.03 %, 2.50 % and 6.88 % were used. The line is a trend-line.
Standard deviations of enzyme loading values as in Figure 1.1.
To further verify the impact of enzyme loading on the packing of cutinase inside the sol-gel
matrices, fluorescence anisotropy decays were measured at different enzyme loadings
(Figure 2.4). The anisotropy fluorescence decays could be fitted by a sum of a one
exponential decay plus a constant that considers the residual anisotropy:
r (t )  1 exp (t /  )   
(3)
Assuming that cutinase is immobilized in the pores, the tryptophan fluorescence anisotropy
results can be interpreted in the context of the wobbling-in-a-cone model which considers
that tryptophan undergoes a restricted non-isotropic motion, wobbling around an axis within
a cone characterized by a certain semi-angle and correlation time, :

r (t )  r (0) x (1  S 2 ) exp( t /  )  S 2

(4)
37
Figure 2.4. : Time resolved anisotropy decay curve, r(t), of the single tryptophan residue of cutinase entrapped
in sol-gel matrices, and the corresponding residual of the fitting by equation 3. A) Matrix with 3.63 % enzyme
loading; B) Matrix with 0.06 % enzyme loading. Standard deviations of enzyme loading values as in Figure
2.1.
The parameter S is a generalized order parameter reflecting the degree of orientational
constraint imposed by the surroundings. If the motion is isotropic, S = 0, and if it is
completely restricted, S=1 (53; 54). Table 2.2. summarizes the results obtained for the fitting of
the tryptophan fluorescence anisotropy decays by equation 3. The anisotropy at time zero is
around 0.3, which is slightly higher than the typical value for tryptophan in solution and in
proteins (~0.25)
(55)
but lower than the theoretical predicted value of 0.4 for collinear
transition dipole moments for absorption and emission. The order parameter S2 is relatively
low, showing that the restrictions imposed by the surroundings are small. This is reasonable
since the tryptophan of cutinase is only partially exposed to its surroundings.
Table 2.2 : Tryptophan fluorescence anisotropy correlation time, θ pre-exponential factor, β1, residual
anisotropy, β∞, and order parameter S2, at different cutinase loadings in the sol-gel matrices.
Enzyme loading %
β1
ps)
β∞
S2
3.63
0.30
2.9
0.022
0.068
1.60
0.34
4.8
0.009
0.029
0.06
0.32
5.5
0.006
0.020
38
Although the order parameter is very small, we have confidence in the values because the
vertical and horizontal polarized decay components where collected alternately with cycles
of 30 s, until the counts in the maximum of the vertical component attained ~200000 counts.
In this way the stability of the system is guaranteed and the accumulation time is rigorously
equal for both components. The almost 4-fold increase in S2 that accompanies the 60-fold
increase in cutinase loading in the sol-gel matrices indicates loss of mobility of the
tryptophan, probably due to protein packing, in agreement with the conclusions drawn on the
basis of the active site titration procedure.
As referred earlier, Reetz and co-workers29 mentioned diffusional limitations of the
substrates as a possible cause for the decrease in specific activity of a sol-gel entrapped
enzyme with the increase in enzyme loading. We are currently studying the diffusion,
through the sol-gel matrix, of relevant species for enzymatic reaction. Here we have focused
on the permeation of the sol-gel matrix by a solvent. The 1H NMR spectrum of
TMOS/BTMS sol-gel is characterized by broad signals due to the residual solid-state effects
related to the dipole-dipole coupling. This line broadening can be substantially reduced by
spinning the sample rapidly around the magic-angle, 54.7° with respect to the z- direction of
the magnetic field. By spinning these heterogeneous samples at the magic angle in a high
rate, anisotropic interactions such as dipolar couplings and magnetic susceptibility
distortions are averaged to their isotropic value, resulting in substantial line narrowing. This
is the basis of the High-resolution magic angle spinning NMR spectroscopy (HR-MAS)
technique that allows the use of high-resolution liquid state NMR pulse programs to study
heterogeneous samples and semi-solid materials 56; 57.
In the present study we have used HR-MAS and pulsed field gradient spin echo experiments
as the analytic method to determine self-diffusion coefficients in the sol-gel matrix. In order
to determine if the presence of the enzyme had any effect on the transport properties of the
matrix, we measured the self-diffusion coefficient of two different solvents in sol-gel
matrices prepared with and without enzyme (Table 2.3). Enzyme loading was set at a value
that already brought about the levelling off of specific enzyme activity.
39
Table 2.3. : Self-diffusion coefficients determined by PFGSEHR-MAS NMR.
Solvent self diffusion coefficient (109 m2/s-1)*
Matrix with 0.97%
Solvent
Matrix without enzyme
enzyme loading
n-hexane
5.04 ± 0.03
4.11 ± 0.26
Methanol
0.82 ± 0.04
0.81± 0.08
*The errors represent the standard deviation of the exponential fitting performed to compute the diffusion.
As can be seen from the table, the values obtained for the cutinase loaded sol-gel matrix and
for the matrix without enzyme are very similar. This shows that the presence of the enzyme
does not impact on solvent diffusion through the sol-gel network and therefore should not
affect the transport of substrates and products. These results thus lend support to the
argument that enzyme aggregation is behind the loss of enzyme specific activity with
increasing enzyme loading.
2.5. Conclusions
We proceeded with our study of the behaviour of cutinase entrapped in sol-gel matrices. We
used different approaches to account for the pronounced increase in the specific activity of
the enzyme with the decrease in enzyme loading, already remarked upon in early studies by
Reetz and co-workers29. The two possibilities put forward then were the aggregation of
enzyme molecules, and the existence of diffusional limitations of the substrates. Active site
titration suggests that cutinase aggregation does occur and its extent parallels the increase in
enzyme loading. Measurements of the fluorescence anisotropy decay of the single
tryptophan residue of cutinase indicate that the increase in enzyme loading is accompanied
by loss of mobility of that residue, which is consistent with enzyme aggregation, in
agreement with the data on active site titration. Self-diffusion coefficients determined by
PFGSE HR-MAS NMR spectroscopy support the hypothesis of enzyme aggregation, since
40
no appreciable difference could be detected for the diffusion of n-hexane and methanol in
enzyme loaded sol-gel matrices and in sol-gel matrices without enzyme.
2.6. Acknowledgments
This work has been supported by Fundação para a Ciência e a Tecnologia through grant no.
PEst-C/EQB/LA0006/2011,
Project
no.
PTDC/QUI/64744/2006,
Grants
no.
SFRH/BD/34800/2007 (GB) and SFRH/BPD/41546/2007 (PV), and by FEDER. The NMR
spectrometers are part of the National NMR Network (RNRMN) and are funded by
Fundação para a Ciência e a Tecnologia.We thank Prof. Luís Fonseca and Mário Fonseca for
the production of cutinase, Marta Corvo for the acquisition of HR-MAS spectra, and Dr.
Alan Phillips for help in measuring enzyme particle sizes.
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40 - Vidinha, P., Barreiros, S., Cabral, J.M.S., Nunes, T.G., Fidalgo, A., Ilharco, L.M. 2008.
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41 - Vidinha P., Augusto V., Nunes, J., Lima, J.C., Cabral, J.M.S., Barreiros S. 2008.
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50 - Petersen, S.B., Fojan, P., Petersen, E.I., Petersen, M.T.V. 2001. The thermal stability
of the Fusarium solani pisi cutinase as a function of pH. J. Biomed. Biotech. 1:2 62–69.
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47
Chapter 3
Thermal stability of sol-gel entrapped
cutinase*
3.1. Abstract
We immobilized cutinase from Fusarium solani pisi in sol–gel matrices of composition 1:5
tetramethoxysilane:n-butyltrimethoxysilane (TMOS/BTMS), at enzyme loadings in the
range of ca. 0.1 % to 4 %, and studied the thermal stability of the enzyme. We found that
submitting the entrapped enzyme to 40 ºC for 24 h had a positive effect on enzyme specific
activity and that thermal treatment for the same length of time at higher temperatures no
longer brought about any activity enhancements. The impact of temperature on enzyme
specific activity was more pronounced in the case of more dilute matrices, whose activity
nonetheless became very similar to that of the more heavily loaded matrices after treatment
at 100 ºC. Steady-state fluorescence measurements did not reveal an increase in fluorescence
emission with exposure to temperature, and time resolved fluorescence showed that the
fluorescence decay times of the single tryptophan of cutinase did not increase as the
temperature of incubation of the sol-gel matrices increased. Both findings indicate that
unlike what happens with cutinase in solution, entrapped cutinase submitted to higher
temperatures does not suffer a denaturation process characterized by ample conformational
mobility in the region of the tryptophan residue. This protective effect is not mediated by
enzyme packing, known to occur at higher enzyme loadings, because not even the more
dilute matrices showed evidence of enzyme denaturation. The matrices are hydrophobic and
have little water. A certain extent of enzyme dehydration upon thermal treatment at moderate
__________________________________________________________________________________
* This chapter has been submitted for publication. Co-authors and affiliations: Gustavo Barreira1, Eurico J.
Cabrita1, Joaquim M.S. Cabral2, José M.G. Martinho3, João Carlos Lima1, Susana Barreiros1;
1
REQUIMTE/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de
Lisboa, 2829-516 Caparica, Portugal; 2IBB-Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia
Biológica e Química, Instituto Superior Técnico, 1049-001, Lisboa, Portugal; 3Centro de Química-Física
Molecular, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
48
temperatures could be beneficial for enzyme conformational mobility and hence enzyme
activity, as observed also in this study, and become detrimental to enzyme function at higher
temperatures. This would explain the similarity of enzyme specific activity values after
thermal treatment at the highest temperature tested, irrespective of enzyme loading.
3.2. Introduction
Cutinase is an enzyme produced by the phytopathogenic fungus Fusarium solani pisi.
Cutinase hydrolyses cutin, an insoluble lipid polyester matrix that covers plant surfaces,
paving the way for infection1 .Cutinase has 197 amino acid residues and is one of the
smallest members of the serine hydrolase family2. Its crystal structure reveals a core formed
by five parallel -strands surrounded by four α-helices. The catalytic machinery of cutinase
comprises the Ser120, Asp175 and His188 triad, and an oxyanion hole2. Cutinase is a
versatile enzyme with several industrial applications, such as laundry and dishwashing
detergent formulations3;4 or agents for the degradation of plastics into water-soluble
products5. Other potential uses include the hydrolysis of milk fat in the dairy industry, the
synthesis of structured triglycerides, polymers and surfactants for personal-care products,
pharmaceuticals and agrochemicals containing one or more chiral centres6 .
The benefits of using enzymes as catalysts at high temperatures, such as an increase in rates
of reactions and yields7, combined with reduced contamination and lower medium
viscosity/facilitated mass transfer8, make thermal stability perhaps the most thoroughly
investigated enzymatic property. The reaction rates of enzyme catalyzed reactions typically
increase exponentially with temperature, until enzyme denaturing becomes prevalent 9. The
most commonly used method to overcome stability issues is the use of immobilization
techniques10. Adsorption onto a solid support is easy to perform, but the binding of the
enzyme to the support is weak. Typically this immobilization technique does not provide a
high degree of stabilization. However, Costa and co-workers11 have found that the
immobilization of cutinase on zeolite NaY has a positive effect on enzyme stability up to 50
ºC, relative to the free enzyme. Covalent attachment to a support often leads to improved
thermal stability. Porous silica is frequently used, but this support does not lead to good
results in the case of cutinase12. Entrapment may have many advantages. Sol-gel
encapsulation consists in weaving a porous network around the enzyme molecules, which act
as templates, so that the nano-capsules thus formed are generally larger than most of the
49
pores prevailing in the gel, in particular in xerogel13. The first reactions to which the sol-gel
precursors are submitted are hydrolysis reactions that lead to the replacement of O-R ligands
by OH ones. Hydrolysis is followed by condensation reactions that lead to the building of a
SiO2 cage around the enzyme molecules.
The use of fluorescence measurements to follow conformational modifications of the
enzymes has become a standard technique to monitor enzymatic stability14;15. Enzyme
fluorescence may have contributions from three types of aromatic residues – tyrosine,
tryptophan and phenylalanine – but typically the phenylalanine quantum yields are too low
to be detected16 . Tyrosine residues emit around 303 nm in aqueous environment and have a
low sensitivity to changes in the local polarity conditions. On the other hand, tryptophan
residues are very suitable probes to assess the conformational state of enzymes, since their
aqueous emission peak is highly dependent on the polarity conditions of the surroundings. In
the native state, tryptophan residues are usually buried in a hydrophobic microenvironment.
When the enzyme unfolds and the tryptophan residues become exposed to a higher polarity
microenvironment, the fluorescence emission intensity maximum is shifted to higher
wavelengths (red shift). In the case of cutinase, which possesses six tyrosine residues
dispersed around the active site and one tryptophan residue located in the opposite region,
the intrinsic fluorescence is dominated by the tyrosine residues, rather than by the
tryptophan. This is due to the disulfide bond located ca. 5Å away from the tryptophan
residue (Trp-69) that is likely to quench the respective fluorescence17; 18. When cutinase is
denatured, the tryptophan residue is exposed to the solvent and becomes dominant in the
light emission spectrum. Recording fluorescence spectra of immobilized cutinase at
increasingly higher temperatures can provide insight into the conformational changes that
occur over the course of the denaturing process19. This has been done for free cutinase and
cutinase immobilized onto several supports14; 19.
In a recent study (chapter 2) we showed that the increase in cutinase loading in the sol-gel
matrices was accompanied by decreases in the specific activity of the enzyme, in the number
of catalytically available active sites, and in tryptophan mobility, which was consistent with
the occurrence of enzyme aggregation in the more heavily loaded matrices. We now focus on
the thermal stability of the entrapped enzyme, and use both steady-state and time resolved
fluorescence to throw insight on the conformational modification of the immobilized
cutinase molecules in response to exposure to high temperatures, for different levels of
enzyme loading within the sol-gel supports.
50
3.3. Materials and methods
3.3.1. Materials
Fusarium solani pisi cutinase was produced by an Escherichia coli WK-6 (a gift from
Corvas International, Ghent, Belgium) and purified at Centro de Engenharia Biológica e
Química, Instituto Superior Técnico44;45. The enzyme purity was controlled by
electrophoresis and isoelectric focusing. (R,S)-2-phenyl-1-propanol (97 % purity), (R,S)-2phenyl-1-propyl butyrate (98 % purity) and tetramethoxysilane (TMOS) were from Aldrich,
n-butyltrimetoxysilane (BTMS) was from Polysciences Inc., vinyl butyrate (99 % purity)
was from Fluka, n-hexane and tridecane were from Merck, Hydranal Coulomat A and C
Karl-Fischer reagents were from Riedel de Häen, polyvinyl alcohol (PVA; MW 15.000) was
from Sigma.
3.3.2. Cutinase immobilization in sol-gel
Following Vidinha and co-workers21 a typical procedure consisted in preparing separately, in
eppendorfs, an aqueous solution containing the enzyme (a given amount of lyophilized
cutinase added to 58 L of 1 M NaF solution, 116 L of 4 % w/v PVA solution and 265L
of distilled-deionised water, making a total of 24.36 mmol of water) and a mixture of
precursors (76 L of TMOS and 487 L of BTMS, to yield a TMOS:BTMS molar ratio of
1:5 and a water/silane molar ratio of 8), adding the latter to the former, under vigorous
shaking on a vortex mixer, until the mixture became homogeneous. The content of the
eppendorf was poured rapidly onto a parafilm-covered plastic tube. The tube was placed in
an ice bath, and kept there for 10 minutes while gelation took place, after which it was kept
at 4 ºC for 24 h. The parafilm holding the material was then placed on a glass dish for air
drying at 35 ºC for 24 h. The resulting xerogel was fragmented and put in an eppendorf that
was placed inside a high pressure cell and submitted to an atmosphere of supercritical CO2 at
40 ºC and 100 bar, for 6 h. The resulting material was left to age at ambient conditions for 5
days before being ground in a mortar, assayed for the amount of immobilized protein, as
determined by a modified Lowry method22 involving pre-treatment with concentrated NaOH
solution at high temperature to release the protein from the matrix, and used for reaction. To
prepare sol-gel matrices with lower enzyme loadings, the above procedure was slightly
51
modified: lyophilized enzyme was replaced by the required amount of enzyme dissolved in
264 L of water. No loss of xerogel material was associated to events other than the removal
of fluids upon drying. The reproducibility of the above procedures for preparing sol-gel
matrices was confirmed by the good agreement between measurements of the amount of
immobilized protein, irrespective of enzyme loading. It was verified that keeping samples at
ambient conditions for up to a month did not impact significantly on enzyme activity.
Enzyme particle sizes were determined for three different enzyme loadings and found not to
depend on the latter parameter, averaging 12040m. The water content of sol-gel matrices
was measured by Karl-Fischer titration, and was usually below 1 % (w/w).
3.3.3. Thermal treatment
Enzyme-loaded sol-gel matrices were submitted to two types of thermal treatment. One
involved increasing the temperature of incubation from 22 ºC to 100 ºC incrementally, at 5
ºC jumps, and holding for 10 min at every temperature plateau. Sample were allowed to cool
down and either assayed for enzyme activity at room temperature, or submitted to a 2 nd or a
3rd cycle. Another involved incubating the sol-gel preparations at temperatures in the range
40-100 ºC for 24 hours in a dry oven, allowing the samples to cool down and assaying for
enzyme activity at room temperature.
3.3.4. Enzyme activity assays
Reactions were performed in n-hexane, in glass vials (reaction volume of 2 mL) placed in a
constant temperature (22 ºC) orbital shaker set for 400 rpm. The reaction studied was the
transesterification of vinyl butyrate (300 mM) by (R,S)-2-phenyl-1-propanol (100 mM). The
addition of the ester marked the start of reaction. The substrates and the solvent were dried
with molecular sieves. Tridecane (20 mM) was used as external standard for GC analysis.
3.3.5. Transesterification reaction analysis
The reaction conversion was measured by GC analysis performed with a Trace 2000 Series
Unicam gas chromatograph equipped with a 30m x 0,32 mm i.d. fused silica capillary
column coated with a 0.25 µm thickness film of 20% 2,3-dimethyl-6-tert-butyldimethylsilyl-
52
β-cyclodextrin dissolved in BGB-15, from Analytik AG. Oven temperature program: 60-180
ºC ramp at 4 ºC min-1, 180-220 ºC ramp at 10 ºC min-1, and holding at 220 ºC for 5 min.
Injection temperature: 250 ºC. Flame ionization detection (FID) temperature: 250 ºC. Carrier
gas: helium (2.0 cm3 min-1). Split ratio: 1:20. No products were detected in assays carried out
without enzyme. The initial rates given (per mg of protein) are the average of at least two
measurements.
3.3.6. Fluorescence anisotropy decays
Steady-state fluorescence spectra of sol-gel samples were recorded on a Cary-Eclipse
fluorescence spectrometer from Varian Inc. with a xenon flash lamp to select the excitation
and emission wavelength. The equipment included a temperature control unit. The excitation
was at 295 nm and the emission fluorescence was recorded from 300 nm to 410 nm. Slit
widths of 1.5 and 10 nm were used for excitation and to record fluorescence emission,
respectively, a narrow excitation slit being needed to prevent cleavage of the disulfide bridge
near the tryptophan residue of cutinase, as referred elsewhere23. Assays were performed in a
4 mL quartz cuvette (10 mm light path). 2 mg of cutinase loaded sol-gel matrix, previously
incubated at the desired temperature, were placed in the cuvette, and were suspended in 1000
µL of 100 mM phosphate buffer at pH 7.5. Time-resolved pico-second fluorescence
measurements were performed using the single-photon counting timing method with laser
excitation. The setup consisted of a mode-locked Spectra-Physics Vanguard 2000-HM532
Nd:YVO4 diode laser, delivering 2 Wof 533 nm light at a repetition rate of 76 MHz and
pulse duration of ≈12 ps that synchronously pumped a cavity dumped 710-2 dye (rhodamine
6G) laser, delivering 3-4 ps pulses at a repetition rate of 1.9 MHz. The laser light was
frequency doubled using a LBO crystal to obtain laser light of 280 nm used for excitation.
Intensity decay measurements were made by an alternate collection of impulse and decay,
with the emission polarizer set at the magic angle position. Impulse was recorded slightly
away from the excitation wavelength with a scattering suspension. For the decays a cutoff
filter was used, effectively removing all excitation light.
3.4. Results and Discussion
In our earlier work with cutinase (chapter 2) we measured enzyme specific activity after a
number of reutilizations. Our experimental procedure involved washing the enzyme loaded
53
support with fresh solvent, followed by drying for 24 h at 40 ºC. We found that enzyme
activity invariably increased from the 1st to the 2nd utilization. We did not explore this fact at
the time, but hypothesized that exposure to a higher, though moderate temperature, might
have a relaxing effect on enzyme conformation, with a positive impact on enzyme activity.
To try and understand this phenomenon better, we exposed enzyme-loaded sol-gel matrices
to a temperature cycle that would take the enzyme to a temperature known to denature free
cutinase in solution. The specific activity of the enzyme increased approximately two-fold up
to the 2nd cycle (Figure 3.1.), suggesting that the thermal treatment imposed on the entrapped
enzyme made it more fit for catalysis, before it decreased at the end of a 3rd cycle.
The procedure we followed when doing reutilization assays involved keeping the enzyme
loaded sol-gel matrices at 40 ºC for 24 h. This led us to study the effect of incubating the solgel preparations at different temperatures for the same period of time (Figure 3.2.). Our
results indicate that doing this type of thermal pre-treatment had a positive effect on enzyme
activity when temperature did not exceed ca. 40 ºC. Submitting the sol-gel entrapped enzyme
to temperatures of ca. 60 ºC either did not improve enzyme activity relative to the blank
(preparation kept at room temperature), or already affected enzyme activity negatively,
an effect that higher temperatures accentuated. The lower the level of enzyme loading and
correspondingly higher specific enzyme activity, the higher the positive impact of thermal
pre-treatment at 40 ºC (Table 3.1).
Figure 3.1. : Initial rates of sol-gel entrapped cutinase kept at room temperature (blank) and submitted to a
number of temperature cycles (initial and final temperatures of 22 ºC and 80 ºC, 5 ºC increases and holding for
10 min at each temperature plateau). Enzyme loading: gray bars, 1.38 %; white bars, 0.52 %. Enzyme specific
activity was measured in a transesterification reaction performed in n-hexane at room temperature. The
standard deviations of enzyme loading values are of ca. 17 %.
54
Figure 3.2. : Impact of the incubation of enzyme loaded sol-gel matrices at temperatures from 40 to 100 ºC, for
24 h, on enzyme specific activity. Enzyme loading: ,0.08 %; , 0.27 %; ▲, 0.52 %; △, 0.97 %; , 3.65%.
The data points at 20 ºC represent supports kept at room temperature. Enzyme activity was measured in a
transesterification reaction performed in n-hexane at room temperature. The standard deviations of enzyme
loading values range from ca. 17 % for higher enzyme loadings, to ca. 32 % for the two lowest enzyme
loadings.
Table 3.1. : Average increase in enzyme specific activity after incubation of the enzyme loaded sol-gel
matrices for 24 h at 40 ºC, relative to incubation for 24 h at 100 ºC. Enzyme activity was measured in a
transesterification reaction performed in n-hexane at room temperature.
Enzyme loading
Average increase in enzyme specific activity
(% w/w)
(nmol min-1 mg-1/ ºC)
0.08
0.27
0.52
0.97
3.65
3843
1150
671
310
187
The specific activity of cutinase should not depend on enzyme loading. The fact that it does,
as we reported recently (chapter 2) and as evidenced by Figure 3.1. and Figure 3.2. for all
temperatures of incubation, led us to consider the possibility of enzyme aggregation. In fact,
we showed in chapter 2 that as enzyme loading in the sol-gel matrices increased, the number
of available enzyme activate sites decreased, and so did the mobility of the tryptophan
55
residue of cutinase, which is consistent with enzyme aggregation in the more heavily loaded
matrices. How does temperature interfere? Figure 3.2. shows that temperature has a very
pronounced effect on the specific activity of cutinase in the less heavily loaded sol-gel
matrix, but has little impact in the case of the more heavily loaded matrix. This could be due
to a protective effect of the packing against thermal denaturation, i.e. cutinase denatures as
temperature increases when it is immobilized in less heavily loaded matrices, while in more
heavily loaded matrices it does not because enzyme aggregation is more pronounced. To test
this hypothesis and look for evidence of protein unfolding upon thermal treatment, we used
fluorescence spectroscopy.
Costa and co-workers11 have performed steady-state fluorescence measurements for free
cutinase in aqueous solution. They observed that the enzyme unfolded as temperature
increased, with concomitant increases in fluorescence emission, the melting temperature of
cutinase being 58-62 ºC. This would lead us to expect a more notorious increase in
fluorescence with increasing temperature in the matrices where the enzyme suffers a higher
degree of unfolding, i.e. the more dilute ones. We performed steady-state fluorescence
measurements with the sol-gel preparations and, unlike what was observed for free cutinase
in solution, the emission spectra did not show increases in intensity with increasing
temperature, for any of the enzyme loadings. This indicates that the process associated to the
decrease in the catalytic activity of the entrapped enzyme (more pronounced in more dilute
matrices) as it is submitted to increasingly higher temperatures does not involve an ample
conformational change of the catalyst in the region of the tryptophan residue, as observed
upon denaturation in aqueous solution.
These findings were confirmed by time resolved fluorescence as a function of temperature.
The average decay times of cutinase did not change significantly with temperature between
20 and 80 º C, for any of the enzyme loadings tested (Figure 3.3).
56
average decay time/ns
3
2.5
2
1.5
1
0.5
0
20
40
60
80
100
temperature /ºC
Figure 3.3. : Decay times of cutinase entrapped in sol-gel matrices, obtained from the fitting of the
fluorescence decay curves by sums of three exponentials. Enzyme loading: ,0.06 %; ○, 0.49 %; □, 1.6%, ,
3.63 %.The scatter observed for the lowest enzyme loading results from photobleaching due to the higher
measurement times under laser irradiation. The standard deviations of enzyme loading values are of ca. 17 %,
except for the lowest enzyme loading, where it is of ca. 32 %.
The fluorescence decays were multi-exponential, with times of ca. 10 ps, 1 ns and 6 ns. The
pre-exponential factor of the decay time of ca. 1 ns accounted for over 95 % of the
fluorescence decay in the sample with the highest loading and was assigned to the enzyme,
while the other two decay times were assigned to the matrix (confirmed by measurements
with matrices without enzyme). It is clear that the process taking place as the matrix is
heated is not the denaturation that occurs in aqueous solution under similar temperature
conditions, for which an increase in decay times is observed11. As a matter of fact, we
recorded a slight decrease in decay times (≈30%), in the sample with higher enzyme loading,
as usually associated to the increase in non-radiative pathways of fluorophores. In the
matrices with lower enzyme loading, increased exposure times led to some photobleaching
of the samples and the fluorescence of the matrix contributed more importantly to the
measured decays, leading to some scatter in the decay times (Figure 3.3). Nevertheless,
within the temperature range studied, there is no evidence of enzyme denaturation even in
the more dilute matrices, in the sense that we do not see any increase in fluorescence
emission with increasing temperature. This indicates that it is not the packing of the enzyme
molecules that is behind the differences observed in the effect of temperature on enzyme
specific activity for different enzyme loadings, shown in Figure 3.2.
If not through the denaturing of the enzyme, in interplay with enzyme aggregation, how else
could temperature interfere? Another possibility is that a species that is relevant for catalysis
57
exists in low concentrations – and therefore its effect is more pronounced at low enzyme
loadings – and its amount in the sol-gel can be affected by thermal treatment. Water fits into
this image. Cutinase is activated by water up to moderately high water activities 24. The water
content of the sol-gel matrices used in the present work was usually below 1 %. The matrices
used have been characterized by diffuse reflectance infrared Fourier transform (DRIFT)
spectroscopy, and the spectra obtained revealed no traces of water25. The enzyme must have
a certain conformational mobility, as provided by hydration, to be catalytically active.
Therefore, some water must be present in the matrices, even if in low amounts. Its effect on
enzyme activity must be more pronounced at low enzyme loadings, because at those
conditions the numbers of water and enzyme molecules will be more equally balanced, than
at high enzyme loadings, when water may not be available to all of the enzyme molecules.
The role of water could account for the dramatic effect of temperature observed for low
enzyme loadings: when the matrices dehydrate, the specific activity of cutinase in the
matrices that offer a more favorable water:enzyme ratio becomes similar to the specific
activity of cutinase in the more heavily loaded matrices, and ceases to depend on enzyme
loading, as one would expect. This would explain the similarity of residual enzyme specific
activity values after exposure of the sol-gel preparations to 100 ºC for 24 h (figure 3.2.), for
all enzyme loadings. We cannot prove that water is playing the role described above, since
the water content of the sol-gel matrices is very low and it is not possible to measure the
range of relevant water content differences accurately. On the other hand, it seems
counterintuitive that treatment of the matrices at e.g. 80º C and 100 ºC for 24 hours should
not yield more similar enzyme activity results, i.e. we would expect that the extent of
enzyme dehydration would attain similar levels in the two cases, and this does not happen
for any of the enzyme loadings. But water is crucial for enzyme performance. The
availability of water to the enzyme, combined with enzyme packing, could account for the
results shown in Figure 3.2.
When commenting on the increased thermal stability of sol-gel entrapped enzymes, some
authors have referred to the enzyme being trapped within a cage built around it during solgel synthesis13 and a positive correlation between lower enzyme flexibility and improved
thermal stability. This has already been highlighted in an early paper by Reetz and coworkers26, who observed an upward shift of 5 ºC in the maximum activity of an immobilized
lipase. Also Nguyen and co-workers27 reported on incomplete denaturation of an
immobilized creatine kinase at temperatures up to 90 ºC, whereas in solution the melting
temperature of the enzyme was 75 ºC. A comparison between the behavior of sol-gel
58
entrapped cutinase with that of cutinase in aqueous solution indicates that the matrix does
have a protective effect on the protein structure – fluorescence spectroscopy shows no
evidence of enzyme denaturation. However, we will need additional approaches to fully
elucidate why it is so.
3.5. Conclusions
The entrapment of cutinase in sol-gel matrices protects the enzyme against thermal
denaturation. Fluorescence spectroscopy measurements revealed that not even in the more
dilute matrices, with higher specific activity, does the enzyme behave as the free enzyme in
solution, i.e. there is no evidence of ample increases in the conformational mobility of the
tryptophan residue when the enzyme is immobilized within the sol-gel support. However, the
specific activity of the enzyme responds to temperature, the enzyme being activated if it is
treated at moderate temperatures, and becoming progressively less active as temperature
increases, reaching an approximately common level of residual specific activity after thermal
treatment at 100 ºC, irrespective of enzyme loading. Enzyme hydration could be a key factor
to explain this behaviour, its decrease beyond a certain level possibly compromising the
required level of enzyme conformational mobility for efficient catalysis. The matrix itself
could evolve under thermal treatment, thereby affecting its interactions with the enzyme. The
tryptophan residue and the active site of cutinase are located at opposite poles of the enzyme
molecule. A probe closer to the catalytic site might provide useful information on how the
catalytic machinery responds to changes in external conditions. In any case, more work is
needed to fully elucidate the events at microscopic scale that lend increased thermal stability
to sol-gel entrapped cutinase.
3.6. Acknowledgments
This work has been supported by Fundação para a Ciência e a Tecnologia through grant no.
PEst-C/EQB/LA0006/2011,
Project
no.
PTDC/QUI/64744/2006,
Grant
no.
SFRH/BD/34800/2007 and by FEDER. We thank Prof. Luís Fonseca and Mário Fonseca for
the production of cutinase, and Dr. Alan Phillips for help in measuring enzyme particle sizes.
59
3.7. References
1 - Purdy, R.E., Kolattukudy, P.E. 1975. Hydrolysis of Plant Cuticle by Plant Pathogens.
Purification, Amino Acid Composition, and Molecular Weight of Two Isozymes of
Cutinase and a Nonspecific Esterase from Fusarium solani pisi. Biochem.14 (13): 2824 –
2831.
2 - Egmond, M.R., de Vileg, J. 2000. Fusarium solani pisi cutinase. Biochem. 82 (11): 1015
– 1021.
3 - Flipsen, J.A.C., Appel, A.C.M., Van der Hijden, H.T.W.M., Verrips, C.T. 1998.
Mechanisms of Removal of Immobilized Triacylglycerol by Lipolytic Enzymes in a
Sequential Laundry Wash Process. Enzyme Microb.Technol, 23 (3-4): 274 – 280.
4 - Unilever, 1994, Enzyme-containing Surfactant Compositions. US Patent 94, 04771.
5 - Murphy, C.A, Cameron, J.A., Huang, S.G., Vinopal, R.T. 1996. Fusarium
Polycaprolactone Depolymerase is Cutinase. Appl. Environm. Microbiol. 62 (2): 456-460.
6 - Carvalho, C.M.L., Cabral. J.S.M., Aires-Barros, M.R. 1999. Cutinase Stability in AOT
Reversed Micelles: System Optimization Using the Factorial Design Methodology.
EnzymeMicrob. Technol. 24 (8-9): 569–576.
7 - Mozahev, V.V. 1993. Mechanism-based strategies for protein stabilization. Trends
Biotechnol 11 (3): 88–95.
8 - Klibanov, A.M., Ahern, T.J., in: Oxender, D.L., Fox, C.F. (Eds.). 1987. Protein
Engineering. Alan R. Liss Inc., New York, Chapter 19, p. 213.
9 - Peterson, M.E., Daniel, R.M., Danson, M.J., Eisenthal, R. 2007. The Dependence of
Enzyme Activity with Temperature: Determination and Validation Parameters.
Biochem. J. 402 (Pt 2): 331 – 337.
10 – Mateo, C., Palomo, J.M., Fernandez-Lorente, G., Guisan, J.M., Fernandez-Lafuente,
R. 2007. Improvement of enzyme activity, stability and selectivity via immobilization
techniques. Enz. Microb. Technol. 40 (6): 1451 – 1463.
60
11 - Costa, L., Brissos, V., Lemos, F., Ramôa-Ribeiro, F., Cabral, J.S.M. 2009. Enhancing
the Thermal Stability of Lipases Through Mutagenesis and Immobilization on Zeolites.
Bioprocess Biosyst. Eng. 32 (1): 53 – 61.
12 - Carvalho, C.M., Aires-Barros, M.R., Cabral. J.M.S. 1999. Cutinase: From Molecular
Level to Bioprocess Development. Biotech. Bioeng. 66 (1): 17 – 34.
13 - Pierre, A.C. 2004.The Sol-Gel Encapsulation of Enzymes. Biocatal. Biotransf. 22 (3),
145 – 170.
14 - Baptista, R.P., Santos, A.M., Federov, A., Martinho, J.M.G., Pichot, C., Elaïssari, A.,
Cabral, J.S.M., Taipa, M.A. 2003. Activity, Conformation and Dynamics of Cutinase
Adsorbed on Poly(methylmetacrylate) Latex Particles. J. Biotecnhol. 102: 241 – 249.
15 - Guo, Q., Zhao, F. Guo S-Y., Wang X. 2004. The Tryptophane Residues of Dimeric
Arginine Kinase: roles of Trp-208 and Trp-218 in Active Site and Conformation
Stability. Biochemie 89 (6): 379 – 386.
16 - Lackowicz, J.R. 2006. Principles of Fluorescence Spectroscopy. 3rded. New York,
Springer, pp. 353-354, 361-364.
17 - Martinho, J.M.G., Santos, A.M., Federov, A., Baptista, R.P, Taipa, M.A, Cabral, J.S.M.
2003. Fluorescence of the Single Tryptophan of Cutinase: Temperature and pH Effect
on Protein Conformation and Dynamics. Photochem.Photobiol.78 (1): 15 – 22.
18 - Prompers, J.J., Hilbers, C.W., Peperman, H.A.M., 1999. Tryptophan mediated
Photoreduction of Dissulfide Bond Causes Unusual Fluorescence Behaviour of
Fusarium solani pisi Cutinase. FEBS Lett, 456 (3): 409 – 416.
19 - Santos, A.M., Federov, A., Martinho, J.M.G. 2008. Orientation of Cutinase Adsorbed
onto PMMA Nanoparticles Probes by Tryptophan Fluorescence. Am. Chem. Soc. 112
(12): 3581 – 3585.
20 - Lauwereys, M., de Geus, P., de Meutter, J., Stanssens, P., Matthyssens, G. 1991. in
Lipases: Mechanism and Genetic Engeneering (Alberghina, L., Schimd, R.D., Verger, R.,
Eds.) Vol. 16, pp 243-251, VCH, Weinheim, Germany.
21 - Vidinha, P., Augusto, V., Almeida, M., Fonseca, I., Fidalgo, A., Ilharco, L.M., Cabral,
J.M.S., Barreiros, S. 2006. Sol-gel encapsulation: An efficient and versatile
61
immobilization technique for cutinase in non-aqueous media. J.Biotecnol. 121 (1): 23 –
33.
22 - Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall. R.J. 1951. Protein measurement
with the Foullin phenol reagent.. J. Biol. Chem. 193: 265-275.
23 - Neves-Petersen, M.T., Gryczynski, Z., Lakowicz, J., Fojan, P., Pedersen, S., Petersen,
E., Bjørn Petersen, S. 2002. High Probability of Disrupting a Disulfide Bridge Mediated
by an Endogenous Excited Tryptophan Residue. Prot. Sci. 11 (3): 588-600.
24 – Garcia, S., Lourenço, N.M.T., Lousa, D., Sequeira, A.F., Mimoso, P., Cabral. J.M.S.,
Afonso, C.M.A., Barreiros, S. 2006. A comparative study of biocatalysis in nonconventional solvents: Ionic liquids, supercritical fluids and organic media. Green
Chem, 6 (9): 466-470.
25 - Vidinha, P., Barreiros, S., Cabral, J.M.S., Nunes, T.G., Fidalgo, A., Ilharco, L.M. 2008.
Enhanced Biocatalytic Activity of ORMOSIL-Encapsulated Cutinase: The Matrix
Structural Perspective. J.Phys. Chem. C. 112 (6): 2008 – 2015.
26 - Reetz, M.T. 1997. Entrapment of Biocatalysts in Hydrophobic Sol-Gel Materials for
Use in Organic Chemistry. Adv. Mater. 9 (12): 943 – 953.
27 - Nguyen, D.T., Smit, M., Dunn, B., Zink, J.I. 2002. Stabilization of Creatine Kinase
Encapsulated in Silicate Sol-Gel Materials and Unusual Temperature Effects on Its
Activity. Chem Mater. 14 (10): 4300 – 4306.
62
Chapter 4
Solvent mobility in sol-gel matrices as
measured by HR-MAS PFGSE NMR
spectroscopy*
4.1. Abstract
Sol-gel entrapment has led to remarkable improvements in enzyme performance in organic
solvents. This fairly stable form of enzyme immobilization prevents unfolding and
denaturation and has led to important improvements when concerning enzyme performance
in organic solvents. Cutinase from Fusarium solani pisi when immobilized in sol-gel
matrices prepared from different combinations of Si(OMe)4 (TMOS) and n-alkyl-Si(OMe)3
(n-alkyl-TMS: M-methyl; P-propyl; B-butyl; OC-octyl) has its highest enzymatic activity in
1:5 TMOS/BTMS sol-gel matrices. In this study we compare the displacement of 2F1P and
water dissolved in different solvents (ACN-aw 0.2, ACN-aw 0.7, n-hexane and methanol) and
of the solvent itself, in TMOS/BTMS sol-gel, in order to determine if this mobility can be
correlated to the observed activity of encapsulated cutinase in the same solvent systems. The
molecular displacement was measured in terms of the self-diffusion coefficient, as
determined by High Resolution Magic Angle Spinning Pulsed Field Gradient Spin Echo
NMR spectroscopy measurements. The solvent self-diffusion was also determined in the
absence of the matrix in order to account for differences in solvent viscosity. Our results
show that diffusion of the solvent through the sol-gel matrix is highly influence by the
chemical nature of the solvent and the interactions with the sol-gel.
_________________________________________________________________________________
* This chapter is being prepared for submission.
63
A model similar to that used in chromatography involving molecular exchange between
different diffusion domains (pore diffusion and surface diffusion) is proposed to rationalize
the experimental results and the mass transfer process.
4.2. Introduction
The encapsulation of bio-molecules in silica sol-gels prepared by hydrolytic polymerization
of tetraethoxysilane has been used as a straightforward procedure for the immobilization of a
variety of interesting molecules1. When concerning enzymes, since these are added at the
early stage of silicate formation, the sol-gel approach results in a fairly stable form of
enzyme immobilization. Because the biocatalysts are trapped within a silica cage, tailored to
their size and shape4 , the mobility ofthe enzyme molecules in the confined space is
restricted, and can prevent unfolding and denaturation2. Sol-gel entrapment has led to
remarkable improvements in enzyme performance in organic solvents3, as evidenced by the
encapsulation of lipases4or a structurally related lyase5 in which rate enhancements of 2-8
fold were registered, compared with the free enzymes. The morphologies of the silica solgels are highly dependent on the method of drying6. When alkylsiloxanes RSi(OR)3 are used
together with Si(OR)4, the surface of the sol-gel is more densely populated by the
hydrophobic alkyl groups, and the capillary stresses that operate on the entrapped enzymes
during evaporation are largely attenuated7. We previously studied the effect of sol-gel
precursors on the catalytic activity of the enzyme cutinase, immobilized in sol-gel matrices
prepared from different combinations of Si(OMe)4 (TMOS) and n-alkyl-Si(OMe)3 (n-alkylTMS: M-methyl; P-propyl; B-butyl; OC-octyl), and showed that enzymatic activity was
highest in 1:5 TMOS/BTMS sol-gel matrices8. It has been suggested that in this matrix the
kinetics of the catalyzed transesterification in n-hexane would be a diffusion-controlled
process9. Therefore the kinetic response of the entrapped biocatalysts would depend on the
substrate diffusion through the porous matrix. Also, the release kinetics of the entrapped
product molecule would also be controlled by its diffusion through the porous sol–gel
particles. The particle morphology of such sol-gel-made materials can be controlled by their
method of preparation, and hence their release characteristics can be tuned. Thus the
knowledge of the pore diffusion coefficient is crucial to understand the transport properties
of molecules within these porous systems. Hence in this study the diffusion coefficients of n-
64
hexane, acetonitrile and methanol were determined by pulsed field gradient NMR
spectroscopy in order to monitor the accessibility of the reactants having in mind the
ultimate comparison of diffusion coefficients of the involved species in the absence of the
sol-gel matrix.
4.2.1. NMR Theory
Pulsed field gradient NMR spectroscopy (PFG-NMR) can be used to observe the
displacement of molecules in a certain period of time during which the molecules will move
to a different position due to self-diffusion. The labelling of the positions of the molecules is
based on the fact that each spin in a magnetic field is characterized by a specific frequency
determined by the magnetic field strength. If a magnetic field gradient whose field strength is
position-dependent is applied then the frequency effect of each spin will also be positiondependent. In a typical PFG-NMR experiment each spin/molecule is firstly labelled
according to its spatial position by a magnetic pulsed field gradient during the gradientencoding period (τ). Then a diffusion time window (Δ) is opened to allow the random
movement of the molecules. Before the NMR signal can be detected, a gradient decode
period (τ) is employed to decrypt the labelling code so that the information about the
molecular movement can be interpreted through the signal attenuation. A series of onedimensional (1D) NMR spectra are then acquired with different gradient values and each
NMR signal (I) decays exponentially as the gradient (q) increases. The I –value is defined as:
I = I0 exp [-q2D (Δ-δ/3)]
(1)
a function of the diffusion coefficient (D), the diffusion time Δ , and the gradient used in the
spatial encoding and decoding of the spin, in which q = γgδ, γ is the gyromagnetic ratio, g is
the gradient strength and δ is the duration over which gradient is applied.
By plotting I versus q2 (Δ-δ/3) and fitting the decay curves, the diffusion coefficient of each
molecule in the system can be determined10.Alternatively the diffusion coefficient can be
obtained from the slope of the plot of ln(I/I0) versus q2 (Δ-δ/3).
65
4.2.2. HR-MAS NMR diffusion spectroscopy
The 1H NMR spectrum of the solvent or substrate molecules in the TMOS/BTMS sol-gel is
characterized by broad signals due to the residual solid-state effects related to the dipoledipole coupling. This line broadening can be substantially reduced by spinning the sample
rapidly around the magic-angle, 54.7° with respect to the z- direction of the magnetic field.
By spinning these heterogeneous samples at the magic angle in a high rate, anisotropic
interactions such as dipolar couplings and magnetic susceptibility distortions are averaged to
their isotropic value, resulting in substantial line narrowing. This is the base of the Highresolution magic angle spinning NMR spectroscopy (HR-MAS) technique that allows the
use of high-resolution liquid state NMR pulse programs to study heterogeneous samples and
semi-solid materials11,12. In this study we have used HRMAS and pulsed field gradient spin
echo experiments as the analytic method to determine self diffusion coefficients in the solgel matrix.
4.2.3. Restricted diffusion
In heterogeneous systems the displacement of the diffusing species depends on interactions
with the porous matrix and may be restricted by pore walls. As the diffusion time increases,
restrictions in the structure are reached and the apparent diffusion rate is decreased. The
longer the molecules diffuse the more restricting barriers will be encountered and as a result,
the measured diffusion coefficient D(t) becomes time-dependent. The long-time behaviour of
D(t) therefore provides an indirect measure of the macroscopic structure.
In the case of restricted diffusion, different regions can be distinguished depending on the
size relations of the barriers, the duration δ of the gradient pulse, and the interval Δ between
the gradient pulses13. Generally in heterogeneous systems, the spin–echo attenuation deviates
from mono-exponential behaviour because various diffusion domains having different
diffusion coefficients may occur. In the case of slow exchange between domains, a multiexponential echo decay will be observed with each domain possessing its own diffusion
coefficient Di
𝐼
𝐼0
𝛿
3
= ∑𝑛𝑖=1 𝑝𝑖 𝑒𝑥𝑝(−𝑞 2 𝐷𝑖 (∆ − ))
(2)
66
with the fraction of molecules in each domain expressed as pi. A diffusion time dependence
of pi can be therefore an indication for an exchange process.
When the exchange between the domains is fast an average diffusion coefficient Dav will be
obtained:
𝐷𝑎𝑣 = ∑𝑛𝑖=1 𝑝𝑖 𝐷𝑖
(3)
and the PFG echo is mono-exponential. Between these two limits different degrees of
exchange may occur between the various domains during the observation time.
4.3. Materials and methods
4.3.1. Materials
The (R,S)-2-phenyl-1-propanol (97 % purity; henceforth referred as 2F1P) and
tetramethoxysilane (TMOS) were from Aldrich, n-butyltrimetoxysilane (BTMS) was from
Polysciences Inc., n-hexane and acetonitrile (99.9 % purity) were from Merck, Hydranal
Coulomat A and C Karl-Fischer reagents were from Riedel de Häen, polyvinyl alcohol
(PVA; MW 15.000) was from Sigma, methanol (99.8 % purity) was from Carlo Erba.
4.3.2. Sol-gel assay preparation
Following Vidinha and co-workers8 a typical procedure consisted in preparing separately, in
eppendorfs, an aqueous solution (58 L of 1 M NaF solution added to 116 L of 4 % w/v
PVA solution and 265L of distilled-deionised water, making a total of 24.36 mmol of
water) and a mixture of precursors (76 L of TMOS and 487 L of BTMS, to yield a
TMOS:BTMS molar ratio of 1:5 and a water/silane molar ratio of 8), adding the latter to the
former, under vigorous shaking on a vortex mixer, until the mixture became homogeneous.
67
The content of the eppendorf was poured rapidly onto a parafilm-covered plastic tube. The
tube was placed in an ice bath, and kept there for 10 minutes while gelation took place, after
which it was kept at 4 ºC for 24 h. The parafilm holding the material was then placed on a
glass dish for air drying at 35 ºC for 24 h. The resulting xerogel was fragmented and put in
an eppendorf that was placed inside a high pressure cell and submitted to an atmosphere of
supercritical CO2 at 40 ºC and 100 bar, for 6 h. The resulting material was left to age at
ambient conditions for 5 days before being ground in a mortar to an average particle size of
12040m, and left overnight submerged in the solutions of interest. Acetonitrile solutions
were prepared by adding distilled-deionised water (1.0 mL and 6.6 mL) to acetonitrile,
keeping a total volume of 100 mL, and aw values were validated by Karl-Fisher titration. The
methanol solution was prepared with 60% (v/v) water content. 2F1P was used at 100 mM in
the various solvents.
4.3.3. NMR assays
All NMR experiments were performed using a Bruker Avance III 400 operating at 400.15
MHz for protons, equipped with a 4 mm high-resolution solid-state Magic Angle Spinning
(MAS) probe and with pulse gradient units, capable of producing magnetic field pulsed
gradients in the z-direction of 0.54 T.m-1. Samples were spun at the magic angle at a rate of 4
kHz, the experimental temperature determined under these conditions was 23 ºC and was
constant within ± 0.1 ºC, as measured using the spectrometer thermocouple system. The
spectra were recorded in 12 L capacity 4 mm ZrO2 rotors. Diffusion measurements were
performed using the stimulated echo sequence using bipolar sine gradient pulses and eddy
current delay before the detection14 .The signal attenuation is given by
æ
æ d ææ
I = I 0 exp æ-g 2 g2d 2 D æD - ææ
æ 3 ææ
æ
(4)
where D denotes the self-diffusion coefficient,  the gyromagnetic ratio,  the gradient pulse
width,  the diffusion time and g the gradient strength corrected according to the shape of
the gradient pulse.Typically, in each experiment a number of 16 spectra of 32K data points
were collected, with values for the duration of the magnetic field pulse gradients (δ) of 1.0 to
2.0 ms, diffusion times (Δ) of 50 to 150 ms and an eddy current delay set to 5 ms. The sine
68
shaped pulsed gradient (g) was incremented from 5 to 95% of the maximum gradient
strength in a linear ramp. The spectra were first processed in the F2 dimension by standard
Fourier transform and baseline correction with the Bruker Topspin software package
(version 2.1). The diffusion coefficients are calculated by an exponential fitting (mono or biexponential) of the data belonging to individual columns of the 2D matrix using Origin 1.2
data software program. Whenever possible the diffusion coefficients (D) were obtained by
measuring the signal intensity at more than one resonance in the spectra. At least two
different measurements were done for the determination of each diffusion coefficient.
4.4. Results and Discussion
Different enzymes encapsulated in sol-gel silica require different degrees of mobility to
perform their catalytic activity. Too much mobility constraint may completely abolish
bioactivity and therefore an understanding of the factors that control the mobility behaviour
of encapsulated guests in sol-gel silica is an important field of research15;16;17. While the
activity and mobility of the encapsulated enzymes has been related to the effects of
Coulombic interactions and physical confinement, there are not many reports concerning the
transport properties of solvent, substracts and/or products in sol-gel matrices and how these
may affect bioactivity.
In this study we compare the displacement of 2F1P and water dissolved in different solvents
(ACN-aw 0.2, ACN-aw 0.7, n-hexane and methanol) and of the solvent itself, in
TMOS/BTMS sol-gel, in order to determine if this mobility can be correlated to the observed
activity of encapsulated cutinase in the same solvent systems. The molecular displacement
was measured in terms of the self-diffusion coefficient, as determined by HRMAS PFGSE
NMR spectroscopy measurements. The solvent self-diffusion was also determined in the
absence of the matrix in order to account for differences in solvent viscosity.
As was mentioned previously the displacement of a diffusing species in a heterogeneous
system, like sol-gel materials, depends on interactions with the porous matrix and may be
restricted by pore walls. The use of pulsed field gradient NMR to measure restricted
diffusion in sol-gel-made porous silica particles has been described previously by Veith and
69
co-workers18. In that study, the restricted diffusion coefficient of water through porous silica
was measured as a function of loading and a model for the water self-diffusion at full pore
filling in sol-gel was derived. The model described the pore or intra-particle water diffusion
as a function of particle porosity, tortuosity, and the steric hindrance applied on the
molecules by the pore space. The intra-particle diffusion coefficient at different degrees of
pore filling was measured as a function of the observation time from bi-exponential fits of
the PFG echo decay. A rapid diffusion regime was found for water for all the observation
times, describing the diffusion according to equation 2, as an exchange between the pores of
the sol-gel matrix and the intra-particle space.
In the work presented in this manuscript the observation time was held constant in all
measurements and the PFGSE decays were analysed as bi-exponential or mono-exponential
decays and compared for the different conditions studied.
4.4.1. Solution self-diffusion coefficients in the absence of matrix
Table 4.1. resumes the self-diffusion coefficients of water and 2F1P in ACN-aw 0.2, ACN-aw
0.7, n-hexane and methanol, as well as the self-diffusion coefficient of the solvents, as
determined in the reference solutions by PFGSE NMR. In these assays, the spin-echo
intensity was observed as a function of the parameter b defined as b = [q2(Δ-δ/3)] and selfdiffusion coefficients were obtained from the slope of the plot ln (I/I0) versus b.
70
Table 4.1. :Self-diffusion coefficient determined by PFGSE NMR technique of water, 2-phenyl-1-propanol
(2F1P), acetonitrile (ACN),methanol (MeOH) and n-hexane in the different reference solutions. Literature
values (Lit) are for the pure solvents, except for methanol.
Diffusion Coefficient (×109 m2.s-1)
Solvent
(ACN, n-hex, or
MeOH)
ACN aw0.2 3.11 ± 0.04
System
Lita
4.3019 ; 3.6520
H2 O
Lit
2F1P
2.74 ± 0.02
-
-
5.08 ± 0.04
-
-
-
-
-
1.27 ± 0.04
1.3724
-
3.99 ± 0.12
-
6.11 ± 0.08
ACN aw0.7
5.25 ± 0.11
n-hexane
7.04 ± 0.06
MeOH 40%
1.11 ± 0.08
ACN aw0.2 +
2F1P
3.38 ± 0.09
1.0324
1.5425
-
ACN aw0.7 +
2F1P
4.11 ± 0.19
-
6.31 ± 0.08
-
4.53 ± 0.10
n-hexane +
2F1P
4.99 ± 0.03
-
-
-
4.21 ± 0.04
MeOH 40% +
2F1P
1.08 ± 0.08
-
1.27± 0.07
-
0.34 ± 0.14
1.2021 ; 3.6022
4.2023
These experimental NMR derived self-diffusion coefficients are similar, or in the same order
of magnitude of the values found in literature, as presented in table 4.1.
For instance MeOH/water solution, self-diffusion values in the 22.87 molar percentage
herein studied was referred by Derlaki24 as 1.03× 10-9m2 s-1 to MeOH and 1.37×10-9 m2 s-1for
water, determined by an experimental technique using a magnetically stirred diaphragm cell
at 25 ºC. Additional self-diffusion coefficient of acetonitrile has been mentioned as 4.3 × 10-9
m2 s-1 (19) or ranging from 0.8 × 10-9 m2 s-1 to 6.5 × 10-9 m2 s-1 (20). The validation was
done however at a more proprietary basis by reference to the NMR technique utilized to
assess the water self diffusion coefficient whose values ranged in between 2,00 ± 0,08 × 10-9
m2 s-1 (27) and 2,025 × 10-9 m2 s-1 (28) at 20 ºC.
71
From the data in table 4.1. is interesting to note that the amount of water in ACN has a
profound influence in the self-diffusion of the acetonitrile. A higher amount of water is
associated with a higher diffusion for acetonitrile either in the absence (3.11× 10-9 m2 s-1 vs
5.25× 10-9 m2 s-1) or in the presence of 2F1P (3.38 × 10-9 m2 s-1vs 4.11× 10-9 m2 s-1). The
same trend is observed for the water (2.74 × 10-9 m2 s-1vs 5.08 × 10-9 m2 s-1in the absence of
2F1P and 3.99 × 10-9 m2 s-1vs 6.31 × 10-9 m2 s-1in the presence of 2F1P) dissolved in the
acetonitrile, a higher amount of water being associated with an overall increase in the selfdiffusion coefficient of water in ACN. This can be explained by a decrease in the viscosity of
the sample upon the increase of water content.
The effect of the addition of 2F1P can also be inferred from this data and seems to be
dependent of the amount of water present. For the sample with 0.2 water activity, both water
and ACN increase their self-diffusion coefficient with the addition of 2F1P, while in the
sample with 0.7 water activity there is an increase in the water self-diffusion but a decrease
in the ACN. Also there is a decrease in the self-diffusion coefficient of 2F1P going from
ACN-aw0.2 to ACN-aw0.7. This may be indicative of an increase in viscosity upon the
addition of 2F1P at aw0.7 or of a process of association of 2F1P under these conditions.
When concerning the influence of the addition of 2F1P in n-hexane or MeOH 40%, the selfdiffusion coefficient values in table 4.1. for the corresponding solvents in the absence and in
the presence of 2F1P show that the effect is minimal in MeOH but that there is an
appreciable decrease in the D of n-hexane with the addition of 2F1P. This last variation is
most probably due to an increase in the solution viscosity.
4.4.2. HRMAS PFGSE NMR self-diffusion coefficients
As was explained above the HR-MAS technique in combination with PFG spin echo
experiments was used to determine the self-diffusion coefficients in the presence of the
matrix. As before, the spin-echo intensity was observed as a function of the parameter b =
[q2(Δ-δ/3)].Echo decays plotted as ln I/I0 versus b for representative data sets for different
conditions are shown in figure 4.1.
72
q2(-/3)
0
0
100000000
200000000
-0,5
300000000
400000000
n-Hexane
water in ACN 0.2w+2F1P
-1
ACN in ACN 0.2w
ln (I/I0)
2F1P in ACN0.2w + 2F1P
-1,5
-2
-2,5
-3
Figure 4.1.: Echo amplitude versus q2(Δ-δ/3) for representative data sets. The PFGSE HR-MAS signal decay
of n-hexane is clearly mono-exponential. Deviations from linearity that evidence multi-exponential behaviour
can be observed for the other datasets.
Deviations from linearity in the plot of ln I/I0 versus b are a clear indication of a multiexponential behaviour. Observation of figure 4.1 clearly shows that depending on the
conditions and on the observed signal, different attenuation behaviours are obtained. A biexponential decay of the echo indicates slow or intermediate exchange between two
diffusion domains, and in these cases two apparent diffusion coefficients can be determined
(Dslow and Dfast) as well as the corresponding normalized fractions (pi according to equation
1). Self-diffusion coefficients as obtained from the experimental data either by mono or biexponential fitting are resumed in Table 4.2. as well as the proportions, pi, in each diffusion
domain in case of bi-exponential fitting.
73
Table 4.2. : Self-diffusion coefficient obtained from the experimental data either by mono or bi-exponential fitting as well as two apparent diffusion coefficients.
Diffusion Coefficient (×109 m2.s-1)
Solvent
Dfast
Dslow
pfast
pslow
ACN aw0.2
10.5 ±
0.12
1.16 ±
0.13
0.68
ACN aw0.7
18.7 ±
0.24
1.92 ±
0.02
0.62
System
Dmono
Water
n-hexane
5.04 ±
0.03
MeOH 40%
0.82 ±
0.43
Dmono
2F1P
Dfast
Dslow
pfast
pslow
0.32
7.01 ±
1.33
0.27 ± 0.11
0.18
0.82
0.38
25.5 ±
18.0
1.22 ± 0.15
0.19
0.81
8.53 ±
3.51
0.50 ± 0.41
0.06
0.94
Dmono
Dfast
Dslow
pfast
pslow
ACN aw0.2 +
2F1P
37.0 ±
0.60
2.28 ±
0.22
0.66
0.34
8.23 ±
2.38
0.45 ± 0.18
0.25
0.75
8.83 ±
4.21
1.70 ±
0.96
0.54
0.46
ACN aw0.7 +
2F1P
7.42 ±
0.74
1.03 ±
0.14
0.71
0.29
7.2± 0.69
0.65E±0.12
0.12
0.88
8.63±1.47
1.47 ±
0.37
0.40
0.60
n-hexane +
2F1P
1.29 ±
0.05
MeOH 40% +
2F1P
1.00 ±
0.18
0.15 ±
0.04
0.409±
0.014
0.46 ±
0.03
74
When concerning restricted diffusion studies, different values for Dslow, Dfast and the
corresponding fractions, pi, are usually obtained by performing different PFGSE NMR
experiments with varying diffusion times. Typically, as the diffusion time is increased a
plateau is reached for the restricted apparent diffusion coefficient in the inter-particle space
because more and more molecules will encounter obstacles. Therefore, by analyzing the
behavior of the determined apparent diffusion coefficients with the observation time
information about the bed packing can be obtained.18
In the present study the diffusion sensitive time interval is kept constant for all the systems
studied and different values for Dslow, Dfast and the corresponding fractions, pi, are obtained,
not as a function of the observation time but as a function of the composition. Under these
conditions, for a fixed observation time, the mono- or bi-exponential behaviour of the echo
decay is due to differences in the fractions of molecules present in the two limiting diffusion
domains and in the exchange rate between these domains. In the limit of slow exchange, the
two limiting diffusion domains can be associated with the diffusion within the pores of the
sol-gel matrix and in the inter-particle space.
The different behaviour found for the systems studied reflect the differences in the partition
constant between the two different diffusion domains for the molecules under observation.
This is ultimately related to specific interactions between the different molecules and the solgel matrix thus making the diffusion experiment a tool to probe these interactions.
4.4.3. Solvent composition affects exchange regime between
diffusion domains
In order to obtain information about the diffusion of individual exchange domains, exchange
constants and residence times, the data presented in table 4.2. must be analysed considering a
model for diffusion in a heterogeneous system with the effects of chemical exchange.
4.4.4. Model for the intermediate regime
A simple scheme to help understand the problem is depicted below, which describes the
diffusion in a heterogeneous system (porous material), where a molecule can adsorb to the
75
surface of the pore where it diffuses more slowly than in the bulk solvent like environment of
the pore.
Pore
diffusion
Surface
diffusion
Solid Phase
S
S
Ds
Dp
k1
SL
Labeled region
L
S
S
SL
k-1
S
Scheme 2- Representation of the diffusion of a substrate (S) molecule in a heterogeneous system under
conditions of two-site exchange. Limiting diffusion environments are represented by DP –diffusion in the pore
and DS –diffusion in the surface; k1 and k-1 represent the exchange rates between the two domains.
This model that takes into account surface phase diffusion is similar to other models
proposed to explain mass transfer in silica gel chromatographic stationary phases.29
The effects of chemical exchange in diffusion spectra have been previously described, and
the exact analytical expressions that describe the magnetization intensity in a diffusion
experiment for a two-site exchange have been derived.30, 31
When the chemical shift difference between the sites is zero or much less than the exchange
rate a single line is observed in the NMR spectrum. Under these conditions the echo decay is
not a simple sum of exponentials because the variable q appears both in the coefficients as
well as in the exponents.
When the exchange between domains is fast compared to the inverse of the diffusion time
(1/Δ), an average diffusion is obtained and the behaviour of the echo attenuation is monoexponential. When the exchange is slow in relation to the inverse of the diffusion time (1/Δ),
the data can be fitted to a bi-exponential and the appropriate diffusion coefficients for each
domain will be observed. In the intermediate exchange regime, the echo decay still reflects
the number of different domains, but the retrieved diffusion coefficients cannot be
straightforwardly assigned to the diffusion in each domain, since they are mixed with the
exchange (partition) rate constants between domains.31
76
If we consider that the molecules adsorbed in the surface layer are immobilized (DS = 0) and
the spin echo decay is essentially from the molecules in the bulk, then the experimentally
observed decay can be used to determine the values of DP and the exchange rates.
The full mathematical treatment of this model is outside the scope of this work, but a
qualitative discussion of the experimental results considering these approximations can be
performed.
The data in table 4.2 show two very distinct behaviours one for n-hexane and MeOH and
another for ACN. The echo decay for n-hexane and MeOH can be described with a single
diffusion coefficient while bi-exponential decay is necessary for ACN. This reflects the fact
that for n-hexane and MeOH the exchange between the two diffusion domains is faster than
that of ACN.
Since the values of the self-diffusion of n-hexane and ACN in the isolated solvents (table 4.1)
are similar, the residence time of n-hexane in the matrix surface must be much less than that
of ACN in order to explain the experimental results in the presence of matrix. The amount of
water in the acetonitrile also affects the apparent diffusion coefficients but the overall biexponential behaviour is maintained. This is not unexpected as the water content was found
to affect the self-diffusion of acetonitrile in the isolated solvents as well (table 4.1) and is
probably due to differences in viscosity.
When considering the water in the ACN water mixtures a bi-exponential decay is also found,
but contrary to the acetonitrile the slow component of the diffusion has a higher contribution
to the overall decay than the fast. This may be indicative of a higher residence time of water
in the surface than that of acetonitrile.
The decay of 2F1P follows the same behavior of the solvent where it is dissolved. In nhexane and methanol the behavior is mono-exponential whereas in ACN it is bi-exponential.
The fact that the 2F1P exchange regime seems to be controlled by the solvent may be
indicative that the molecule is efficiently solvated and that its partition accompanies that of
the solvent.
In general the more polar molecules (ACN and H2O) seem to have a higher residence time in
the surface of the matrix, whereas the apolar n-hexane exchanges fast between the diffusion
domains.
77
4.5. Conclusions
Self-diffusion coefficients of 2F1P and water dissolved in different solvents (ACN-aw 0.2,
ACN-aw 0.7, n-hexane and methanol) and of the solvent itself were determined in a
TMOS/BTMS sol-gel matrix using PFGSE HRMAS NMR. The PFG spin echo decays of the
studied molecules within the sol-gel reflect conditions of restricted diffusion under exchange
conditions. It was found that the diffusional behaviour of the solvent, water or 2F1P is
controlled by an exchange process between to limiting diffusion environments (Dfast and
Dslow). Our results show that solvent composition affects the exchange rate and the partition
between the two limiting diffusional domains. A model taking into account surface phase
diffusion similar to models proposed to explain mass transfer in silica gel chromatographic
stationary phases was proposed to rationalize the results. Solvent composition determines the
type of behaviour found for the systems studied, mono- or bi-exponential echo decay. These
are due to differences in the fractions of molecules present in the two limiting diffusion
domains and in the exchange rate between these domains. When the exchange between
domains is fast just like in the case of n-hexane, MeOH and 2F1P in n-hexane, the behaviour
of the echo attenuation is mono-exponential and a single diffusion coefficient is obtained
from the PFGSE experiment. When the exchange between domains is slow, the data can be
fitted to a bi-exponential and two diffusion coefficients are observed. This is the case of
acetonitrile, and water and 2F1P in acetonitrile. These results indicate that acetonitrile has a
higher residence time in the surface of the sol-gel than n-hexane. On the other hand, the
behaviour of 2F1P seems to be determined by the solvent where it is dissolved and not by
specific interactions with the sol-gel matrix.
A full mathematical analysis of the proposed diffusional model is currently under
development and may be used to determine the values of DP and the exchange rates.
4.6. Acknowledgments
This work has been supported by Fundação para a Ciência e a Tecnologia through grant no.
PEst-C/EQB/LA0006/2011,
Project
no.
PTDC/QUI/64744/2006,
Grant
no.
SFRH/BD/34800/2007 and by FEDER. The NMR spectrometers are part of the National
NMR Network (RNRMN) and are funded by Fundação para a Ciência e a Tecnologia.
78
4.7. References
1 -Avnir, D., Coradin, T., Lev, O., Livage, J. 2006. Recent bio-applications of sol–gel
materials. J. Mater. Chem. 16: 1013-1030.
2 -Gill, I., Pastor, E., Ballesteros, A. 1999. Lipase-silicone Biocomposites: Efficient and
Versatile Immobilized Biocatalysts. J.Am.Chem.Soc.121: 9487 – 9496
3 -Hara, P., Hanefeld, U., Kanerva, L. 2008.
Sol-gels and cross-linked aggregates of
lipase PS from Burkholderia cepacia and their application in dry organic solvents. J.
Mol. Catal. B: Enzymatic. 50: 80-86.
4 -Reetz, M., Tielmenn, P., Wiesenhöfer, W., Könen,W., Zonta, A. 2003. Second generation
sol-gel encapsulated lipases: robust heterogeneous biocatalysts. Adv. Synth. Catal. 354:
717 – 728.
5 -Cabirol, F., Hanefeld, U., Sheldon, R. 2006. Immobilized hydroxynitrile lyases for
enantioselective
synthesis of cyanohydrins: sol-gels and cross-linked enzyme
aggregates. Adv. Synth. Catal.348:1645-1654.
6 -Pierre, A., Pajonk, G. 2002. Chemistry of Aerogels and Their Applications.Chem. Rev
102 : 4243 - 4264
7 -Sheldon R. 2007. Enzyme Immobilization: The Quest for Optimum Performance. Adv.
Synth. Catal.349: 1289 – 1307.
8 -Vidinha, P., Augusto, V., Almeida, M., Fonseca, I., Fidalgo, A., Ilharco, L., Cabral, J.,
Barreiros, S. 2006. Sol-gel encapsulation: An efficient and versatile immobilization
technique for cutinase in non-aqueous media. J.Biotecnol. 121: 23 –
9 -Vidinha, P., Barreiros, S., Cabral, J., Nunes, T., Fidalgo, A., Ilharco, L. 2008. Enhanced
Biocatalytic Activity of ORMOSIL-Encapsulated Cutinase: The Matrix Structural
Perspective. J.Phys. Chem. C. 118: 2008 – 2015.
10 -Johnson, C.S. 1999. Diffusion ordered nuclear magnetic resonance spectroscopy:
Principlesand applications. Prog. Nucl. Magn. Reson.Spectrosc., 34, 203-256.
79
11 - Viel, S., Ziarelli, F., Caldarelli, S. 2003. Enhanced diffusion-edited NMR
spectroscopy of mixtures using chromatographic stationary phases, PNAS,100, 96969698;
12 - Carvalho, L. R., Corvo, M. C., Enugala, R., Marques, M. M. B., Cabrita, E. J. 2010.
Application of HR-MAS NMR in the solid-phase synthesis of a glycopeptide using
Sieber amide resin, Magn. Reson. Chem., 48, 323-330
13 - L.Z. Wang, A. Caprihan, E. Fukushima.1995. The narrow pulse criterion for pulsed
gradient spin echo diffusion measurements.J. Magn. Reson. Ser. A, 117: 209- 219
14 - D. Wu, A. Chen and C. S. Johnson Jr., J. 1995. An Improved Diffusion-Ordered
Spectroscopy Experiment Incorporating Bipolar-Gradient Pulses. J.Magn. Reson., A115,
260-264.
15 - Hanefeld, U., Gardossi, L., Magner, E. 2009. Understanding enzyme immobilization.
Che. Soc.Rev. 38: 453-468.
16 - Sheldon, R. 2007. Enzyme immobilization: the quest for Optimum Performance.
Adv Synth. Catal. 349: 1289 – 1307.
17 - Avnir, D., Croadin, T., Lev, O., Livage, J. 2006. Recent bio-applications of sol-gel
materials. J. Mater. Chem. 16: 1013 – 1030.
18 - Susanne R. Veith, Eric Hughes, Gilles Vuataz, and Sotiris E. Pratsinis. 2004.Restricted
diffusion in silica particles measured by pulsed field gradient NMR. Journal of Colloid
and Interface Science274: 216–228
19 - Raymond, D. 1997. Shear Viscosity and Dialectric Constant of Liquid Acetonitrile.
J. Chem, Phys.107: 3921 – 3923
20 - Hurdle, R., Woolf, L. 1982. Self-Diffusion in Liquid Acetonitrile under Pressure. J.
Chem. Soc. Faraday Trans. 78: 2233 – 2238
21 - Peksa, M., Lang, J., Kocirík, M. 2009. PFG NMR Study of Liquid n-Hexane SelfDiffusion in Bed of Porous Glass Beads. Diffusion-fundamentals.org 11(36) : 1- 2.
80
22 - D’Agostino, C., Mantle, M., Gladden, L., Moggridge, G. 2011. Prediction of binary
diffusion coefficients in non-ideal mixtures from NMR data: Hexane-nitrobenzene near
its consolute point. Chem. Eng. Science, 66: 3989 – 3906.
23 - Iwahashi, M., Kasahara, Y. 2007. Effect of olecular Size and Structure on the SelDiffusion Coefficient and Viscosity for Saturated Hydrocarbons Having Six Carbon
Atoms. Jornal of Oleo Science, 56: 443 – 448.
24 - Derlacki, J., Easteal, A., Edge, A., Woolf, L. 1985. Diffusion Coefficients of Methanol
and Water and the Mutual Diffusion Coefficients in Methanol-Water Solutions at 278
and 298 K. J. Phys Chem. 89: 5318 – 5322.
25 - Rah, R., Kwak, S., Chang Eu, B. Lafleur, M. 2002. Relation of Tracer Diffusion
Coefficient and Solvent Self-Diffusion Coefficient. J.Phys. Chem. 106:11841 - 11845.
26 - Su, J., Ducan, P., Momaya, A., Jutila, A., Needham D. 2010. The Effect of Hydrogen
Bonding on the Diffusion of Water in n-Alkanes and n-Alcohols Measured with a Novel
Single Microdoplet Method. J. Chem. Phys. 132: 044506.
27 - Turanov, A., Yuriy, T. 2009. Turanov, A., Yuriy, T. 2009. Heat- and mass-transport in
aqueous silica nanofluids. Heat Mass Transfer 45: 1583 – 1588.
28 - Holz, M., Heil, S., Sacco, A. 2000. Temperature-dependent Self-diffusion
Coefficients of Water and Six Selected Molecular Liquids for Calibration in Accurate
1H
NMR PFG Measurements. Phys. Chem.Chem. Phys. 2: 4740 – 4742.
29 – Miyabe, K. 2007. Surface diffusion in reversed-phase liquid chromatography using
silica gel stationary phases of different C1 and C18 ligand densities. Journal of
Chromatography A; 1167: 161-170.
30 –Johnson, Jr, C. S. 1993. Effects of chemical exchange in diffusion-ordered 2D NMR
spectra, Journal of Magnetic Resonance A 102: 214-218.
81
31 - Cabrita, E. J., Berger, S., Bräuer, P., Kärger, J. 2002.High-Resolution DOSY NMR
with Spins in Different Chemical Surroundings: Influence of Particle Exchange. Journal
of Magnetic Resonance, 147: 124-131.
82
5. Conclusions
The work developed along this thesis shows that understanding the behaviour of immobilized
cutinase in sol-gel matrices is still a promising and challenging field of research. In the
course of the three article-chapters here included some issues emerged that could be
addressed in further studies. So as corollary of this thesis which, as every other scientific
study, is never quite finished, some lines of work are pointed out that might add to the studies
here described.
The possibility of enzyme aggregation occurring in sol-gel supports was mentioned in 1996
by Reetz and co-workers. Still, neither these authors nor others authors have addressed the
confirmation
of
that
possibility thoroughly.
This
provided
motivation
for
the
multidisciplinary approach used in this thesis, focused on the enzyme cutinase, combining
medium engineering studies with fluorescence and NMR spectroscopy studies. As referred
earlier, cutinase has only one tryptophan residue, which allows the monitoring of
conformational changes of the molecule through fluorescence emission. However, tryptophan
is not located in the catalytic region of the enzyme, but rather opposite it. More accurate
information about structural changes imposed by overloading or thermal exposure with
impact on enzymatic activity would benefit from a probe closer to the enzyme active site.
Building such a probe has been the object of other studies performed within a project aimed
at elucidating cutinase/matrix interactions, to which this thesis represents a contribution.
Mutant N84W (Figure 5.1), with a second tryptophan residue close to the enzyme active site
has been generated and immobilized in sol-gel matrices of different compositions. It showed
much higher fluorescence emission intensity than wild-type cutinase, i.e. the signal from the
second, unquenched tryptophan, dominates over the signal of the native tryptophan. Mutant
N84W exhibits only a fraction of the catalytic activity of native cutinase for substrates with
carbon chains longer than C3, due to spatial constraints imposed by the second tryptophan 1.
Thus, substrates adequate for measuring the catalytic activity of the sol-gel entrapped mutant
should first be assayed with the sol-gel entrapped native enzyme, for calibration. Then the
influence of enzyme loading on enzyme specific enzymatic activity would be determined.
83
Figure 5.1. : N84W cutinase mutant, with additional tryptophan residue shown as sticks (on top). Also shown
as sticks are the residues of the catalytic triad (Ser120, Asp175, H188 ).
The results obtained in this thesis suggest that the enzyme molecules aggregate within the
sol-gel matrices once a certain level of enzyme loading is reached, as evidenced by the active
site titration studies performed. The fluorescence anisotropy decay measurements indicated a
loss of mobility of the tryptophan residue of cutinase in the more heavily loaded sol-gel
matrices, which was consistent with enzyme aggregation. It was also shown that the presence
of the enzyme in the sol-gel matrices, at concentrations that already caused the leveling out of
enzyme specific activity, had a negligible effect on solvent mobility within the matrix. These
three facts suggest additional studies with the more heavily loaded supports, based on the
hypothesis that the clusters of aggregated enzyme molecules, which NMR studies have
shown not to clutter the sol-gel matrix, have internal mobility.
The possibility for such dynamic behavior along the successive catalytic cycles is
strengthened by the fact that exposure of the entrapped enzyme to 40 ºC for 24 h had a
positive effect on enzyme specific activity, suggesting some adaptation of the enzyme to the
structural features of the matrix. This hypothesized mobility could be assessed by studies
with differently loaded supports in the concentration range where evidence of aggregation
exists, submitted to a large number of utilizations and inhibition assays. In this thesis, the
entrapped enzyme was irreversibly inhibited with different inhibitor concentrations, and the
84
matrices were then assayed for residual enzyme activity. If there is a certain degree of
internal molecular mobility in the enzyme agglomerates, then upon reutilization and gentle
thermal treatment, there might be a re-accommodation of the enzyme molecules, with some
molecules at the core of the agglomerate turning to the external surface.
This approach could also be applied to thermal treatment assays. The results obtained in this
thesis show that after exposure of differently loaded sol-gel matrices to 100 ºC, enzyme
specific activity values become very similar. It was hypothesized that water, which plays a
crucial role in enzyme dynamics and activity, existed in the hydrophobic matrices in low
amounts, too low to accommodate the needs of all the molecules present in the more heavily
loaded supports. Upon thermal treatment at 100 ºC, the amount of water remaining in the
matrices would be too low even for the enzyme molecules of the more dilute sol-gel supports.
One question that may arise is what if the matrices submitted to 100 ºC were rehydrated?
They could be rehydrated at different water activity levels, and assayed again. This might
facilitate the hypothesized migration of core molecules of the agglomerates to peripheral
regions, and bring about increases in enzyme specific activity. As mentioned above, a
tryptophan residue closer to the enzyme active site might provide more accurate information
about structural changes occurring upon thermal exposure and help clarify the role of water,
with a view to elucidating the stabilizing effect of entrapment within sol-gel matrices.
The results obtained by NMR on diffusion within the sol-gel matrices led to a model with
two limiting diffusion environments – diffusion in the pore, i.e. diffusion in the bulk solventlike environment of the pore, and diffusion at the surface, i.e. slower diffusion of the
adsorbed molecules at the surface of the pore. All experiments were performed at a fixed
time of observation. By changing the latter parameter, the reference for studying the
phenomenon changes too. When observation times are too short, species cannot diffuse freely
between the two diffusion domains while the measurements are made. When observation
times are long enough, the two diffusion domains become indistinguishable. Further studies
at different times of observation should allow a better characterization of diffusion within the
sol-gel matrices. Also in line with further elucidation of the diffusion phenomenon, studies
with matrices of different compositions should also be useful, e.g. TMOS/MTMS, where the
second precursor bears a methyl group instead of n-butyl, to see how a coordinated change in
polarity of the sol-gel microenvironment might reflect on the mobility of diffusing species
within the matrices.
85
In closing, the high stability of cutinase imposed by its entrapment in TMOS/BTMS sol-gel
matrices, highlighted in this thesis, should lead to a broad range of useful applications.
5.1. Reference
1 - Nicolas, A., Egmond, M., Verrips, C., Jakob de Vlieg, Longhi, S., Cambillau, C.,
Martinezz, C. 1996. Contribution of Cutinase Serine 42 Side Chain to the Stabilization of
the Oxyanion Transition State. Biochemistry, 35: 398-410.
86