Luís Gustavo Godinho Barreira
Mestre em Parasitolgia Médica
C
HARACTERIZATION OF SOL-
GEL MATRICESWITH 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
“Science should be as simple as possible but not simpler”:
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.
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, recorreu-se à titulação de centros activos, barecorreu-seada 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 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 propõe-semelhante ao que é utilizado em cromatografia, envolvendo permuta de moléculas entre diferentes domínios difusionais (difusão nos poros e difusão à superfície).
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 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 self-diffusion 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.
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
ACN - acetonitrile aW – water activity
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
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 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...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
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
Tables Index
Table 1.1. : Lipase loadings and relative amounts of immobilized enzyme in sol-gel matrices. ...7XFigure 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,
2-phenyl-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,
2-phenyl-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
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 fine-chemicals, 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
applications as biomedical products5, products for the treatment of intolerance to lactose6, artificial organs design7, antibiotics production8;9, biomass hydrolysis10, bioremediation11;12;13, chemical sensors14;15, biosensors16;17;18;19, 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
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 Si-OH 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
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
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
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 alkyltrimethoxysilane (alkylTMS) in the order methyl < ethyl < propyl < n-butyl. 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).
Table 1.1. : Lipase loadings and relative amounts of immobilized enzyme in sol-gel matrices.
Enzyme Enzyme loading
Enzyme retained from 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).
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.
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 (31; Cys-109) 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 breath-like 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 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 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.
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 29Si 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 C
4 – 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 charges44 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 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 residues48 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 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 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.
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.
1.4. References
1- Hanefeld, U., Gardossi, L., Magner, E. 2009. Understanding enzyme immobilization. Che. Soc.Rev. 38: 453-468.
2- Sheldon, R. 2007. Enzyme immobilization: the quest for Optimum Performance. Adv Synth. Catal. 349: 1289 – 1307.
3- Avnir, D., Croadin, T., Lev, O., Livage, J. 2006. Recent bio-applications of sol-gel
materials. J. Mater. Chem. 16: 1013 – 1030.
4- Kim, J., Grate, J., Wang, P. 2006. Nanostructures for enzyme stabilization. Chem. Eng. Science 61: 1017 – 1026.
5 - Chiriac, P., Neamtu, I., Nita, L., Nistor, M. 2010. Sol Gel Method Performed for
Biomedical Products Implementation. Bentham Sc. Publis., Mini Reviews in Medicinal Chemistry 10: 990 – 1013.
6 - Nichele,V., Signoretto, M., Ghedini, E. 2011. β-Galactosidase entrapment in silica gel
matrices for a more effective treatment of lactose intolerance. J. Mol. Catal. B: Enz. 71 :
10-15.
7 - Dandoy, P., Meunier, C., Michiels, C., Su, B. 2011. Hybrid Shell Engineering of
Animal Cells for Immune Protections and Regulation of Drug Delivery: Towards the Design of “Artificial Organs”. Plos One 6: Article number: e20983.
8 - Bernardino, S., Fernandes, P., Fonseca, L. 2010. Improved specific productivity in
cephalexin synthesis by immobilized PGA in silica magnetic micro-particles. Biotechnol
Bioeng. 107: 753–762.
9 - Bernardino, S., Estrela, N., Ochoa-Mendes, V., Fernandes, P., Fonseca, L. 2011.
Optimization in the immobilization of penicillin G acylase by entrapment in xerogel particles with magnetic properties. J. Sol-Gel Sci. Techn. 58: 545–556.
10 - Das, S., Berke-Schlessel, D., Ji, H., McDonough, J., Wei, Y. 2011. Enzymatic
hydrolysis of biomass with recyclable use of cellobiase enzyme immobilized in sol–gel routed mesoporous silica. J. Mol. Catal. B: Enzym.70 : 49-54
11 - Sani, S., Muhid, M., Hamdan, H. 2011. Design, synthesis and activity study of
tyrosinase encapsulated silica aerogel (TESA) biosensor for phenol removal in aqueous solution. J.Sol-Gel Sci. Techn. 59: 7 – 18.
12 - Li, Y., Zhang, H., Cao, F. 2011. Analysis of phenolic compounds catalyzed by
13 - Oriero, D., Jabal, J., Deobald, L., Weakley, A., Aston, D. 2011. A potential
enzyme-encapsulating, ultrafine fiber for phenol detection. Reac. Funct. Polym . 71: 870-880. 14 - Tran-Thi, T., Dagnelie, R., Crunaire, S. Nicole, L. 2011. Optical chemical sensors
based on hybrid organic–inorganic sol–gel nanoreactors. Chem. Soc. Rev. 40: 621-639.
15 - Wu, M., Wu, R., Zhang, Z., Zou, H. 2011. Preparation and application of
organic-silica hybrid monolithic capillary columns. Electrophoresis 32:105-115
16 - Parra-Alfambra, A., Casero, E., Petit-Domínguez, M., Barbadillo, M., Pariente, F., Vázquez, L., Lorenzo, E. 2011. New nanostructured electrochemical biosensors based on
three-dimensional (3-mercaptopropyl)-trimethoxysilane network. Analyst. 136: 340-347.
17 - Jena, K., Raj, R. 2011. Enzyme integrated silicate-Pt nanoparticle architecture: a
versatile biosensing platform. Biosens Bioelectron . 26: 2960 – 2966.
18 - Ozturk, G., Feller, K., Dornbusch, K., Timur, S., Alp, S., Ergun, Y. 2011. Development of Fluorescent Array Based on Sol-Gel/ Chitosan Encapsulated Acetylcholinesterase and pH Sensitive Oxazol-5-one Derivative. J. Fluoresc. 21: 161 – 167.
19 - Zhang, L., Wang, G., Xing, Z. 2011. Polysaccharide-assisted incorporation of
multiwalled carbon nanotubes into sol–gel silica matrix for electrochemical sensing. J.
Mater. Chem. 21: 4650-4656.
20 - Fu, G., Yue, X., Dai, Z. 2011. Glucose biosensor based on covalent immobilization of
enzyme in sol-gel composite film combined with Prussian blue/carbon nanotubes hybrid. Biosens Bioelectron.9: 3973 – 3976.
21 - Rosario, E., Ventura, B., Nicola, S., Altucci, C., Velotta R., Mita, D., Lepore, M. 2011.
Glucose Sensing by Time-Resolved Fluorescence of Sol-Gel Immobilized Glucose Oxidase. Sensors, 11: 3483-3497
22 - Zamfir, G., Rotariu, L., Bala C. 2011. A novel, sensitive, reusable and low potential
acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel. Biosens. Bioelectron . 26: 3692-3705.
23 – Rassy, H., Perrard, A., Pierre, A. 2003. Behaviour of Silica Aerogel Network as
Highly Porous Solid Solvent Media for Lipases in a Model Transesterification Reaction. Chem. Biochem. 44: 203 – 210.
24 - Livage, J., Henry, M. and Sanchez, C. 1988. Sol-gel chemistry of transition metal
oxides. Prog. Sol. State Chem. 18: 259 – 341.
25 – Pierre, A. 2004. The sol-Gel Encapsulation of Enzymes. Biocatal Biotransformation
26 - Gill, I. 2001. Bio-doped nanocomposite polymers: sol-gel bioencapsulates. Chem. Mat. 13: 3404 - 3421.
27 - El Rassy, H., Buissin, P., Bouali, B., Perrard, A., Pierre, A. 2003. Surface
Characterization of Silica Aerogel with Different Proportions of Hydrophobic Groups, Dried by CO2 Supercritical Method. Langmuir,19, 358 – 363.
28 - Reetz, M., Zonta, A., Simplekamp, J., Konen, W. 1996. In situ fixation of
lipase-containing hydrophobic sol-gel materials on stirred glass-highly efficient heterogeneous biocatalyst. Chem. Comm. 11: 1397 – 1398.
29 - Reetz, M. 1997. Entrapment of Biocatalysts in Hydrophobic Sol-Gel Materials for
Use in Organic Chemistry. Adv Mater., 9, 943-953.
30 - Reetz, M., Tielmann, P., Wiesenhöfer, W., Könen, W., Zonta, A. 2003. Second
Generation Sol-Gel Encasuplated Lipases: Robust Heterogeneous Biocatalysts. Adv.
Synth. Catal. 345, 717 – 728.
31 - Pierre, A., Pajonk, G. 2002. Chemistry of aerogels and their applications. Chem. Rev.
102: 4243 - 4265.
32 - Baker, G., Jordan, J., Bright, F. 1998. Effects of Poly(ethylene glycol) doping on the
behavior of Pyrene, Rhodamine 6G, and acrylodan-labeled bovine serum albumin sequestered within tetramethylorthosilane-derived solgel-processed composites. J.
Sol-Gel Sci. Technol. 11: 43 - 54.
33 - Bergogne, L., Fennouh, S., Guyon, S., Roux, C., Livage, J. 2001. Sol-gel entrapment
of enzymes. Materials Research Society Symposium Proceedings 628: (Organic/Inorganic
Hybrid Materials) CC10.2.1 - CC10.2.6.
34 - Samuni, U., Navati, M., Juszcsak, L., Dantsker, D., Yang, M., Friedmanm J. 2000.Unfolding and Refolding of Sol-gel Encapsulated Carbonmonoxymyoglobin: an
Orchestrated Spectroscopy Study of Intermediates and Kinetics. J.Phy. Chem. B., 104,
10802 – 10813.
35 - Zhou, Y., Dill, K. 2001. Stabilization of Proteins in Confined Spaces. Biochemistry,
40, 11289 – 11293.
36 - Bismuto, E., Martelli, P., De Maio, A., Mita Damiano, G., Irace, G. 2002. Effect of
Molecular Confinement on Internal Enzyme Dynamics: Frequency Domains Fluorometry and Molecular Dynamics Simulation Study. Biopolymers, 67(2), 85 – 95.
37 - Nguyen, D., Smit, M., Dunn, B., Zink, J. 2002. Stabilization of Creatine Kinase
Encapsulated in Silicate Sol-Gel Materials and Unusual Temperature Effects on Its Activity. Chem Mater. 14, 4300 – 4306.
38 - Badjiç, D., Kostiç, M., 1999. Effects of Encapsulation in Sol-gel Silica Glass on Esterase Activity, Conformational Stability, and Unfolding of Bovine Carbonic Anydrase II. Chem.
Mater. 11(12), 3671 – 3679.
39 - Flora, K., Brennan, J., Baker, G., Doody, M., Bright, F. 1998. Unfolding of
acrylodan-labeled human serum albumin probed by steady-state and time-resolved fluorescence methods. J. Biophy. 75: 1084 -1096.
40 - Zheng, L., Brennan, J. 1998. Measurement of intrinsic fluorescence to probe the
conformational flexibility and thermodynamic stability of a single tryptophan protein entrapped in a sol-gel derived glass matrix. Analyst 123: 1735 - 1748.
41 - Altstein, M., Segev, G., Aharonson, N., Ben-Aziz, O., Turniansky, A., Avnir, D. 1998.
Sol-gel-entrapped cholinesterases: a microtiter plate method for monitoring anti-cholinesterase compounds. J. Agric. Food. Chem. 46: 3318 - 3324.
42 - Wambolt, C.L. and Saavedra, S. 1996. Iodide fluorescence quenching of sol-gel
immobilized BSA. J. Sol-Gel Sci. Tech. 7: 53 - 57.
43 - Brennan, J., Benjamin, D., DiBattista, E., Gulcev, M. 2003. Using Sugar and Amino
Acid Additives to Stabilize Enzymes within Sol-Gel Derived Silica. Chem. Mater, 15, 737
– 745.
44 - Maury, S., Buisson, P., Perrard, A., Pierre, A. 2005. Compared Esterification Kinetics
of the Lipase from Burkholderia cepacia either free or encapsulated in a silica aerogel.
J. Mol. Catal B Enz 32, 193-203.
45 - Bathia, B., Brinker, C., Gupta, A., Singh A. 2000. Aqueous Sol-gel Proces for Protein
Encapsulation. Chem. Mat., 12, 2434 – 2441.
46 - Chen, J., Wang, H. 1998. Improved properties of bilirubin oxidase by entrapment in
alginate-silicate sol-gel matrix. Biotechnol. Tech. 12: 851 - 853.
47 - Noureddini, H., Gao, X., Joshi, S., Wagner, P. 2002. Immobilization of Pseudomonas
cepacia Lipase by Sol-Gel Entrapment and Its Application in the Hydrolysis of Soybean
Oil. J. Am. Oil Chem. Soc., 79(1), 33 – 40.
48 – Santos, A., Federov, A., Martinho, J. 2008. Orientation of Cutinase Adsorbed onto
PMMA Nanoparticles Probed by Tryptophan Fluorescence. Am. Chem. Soc. 112: 3581 –
3585.
49 - Chen, J., Lin, W. 2003. Sol-gel powders and supported sol-gel polymers for
immobilization of lipase in ester synthesis. Enzy. Mic. Technol. 32(7): 801 - 811.
50 - Vidinha P., Augusto V., Almeida M., Fonseca I., Fidalgo A., Ilharco L., Sobral J., Barreiros S. 2006. Sol-gel encapsulation: An efficient and versatile immobilization
51 - Gill, I., Ballesteros, A. 1998. Encapsulation of biological within silicate, siloxane,
and hybrid sol-gel polymers: an efficient and generic approach. J. Am. Chem. Soc. 120:
8587 - 8598.
52 - Gill, I., Ballesteros, A. 2000. Bioencapsulation within synthetic polymers (Part 1):
sol-gel encapsulated biological. Trends Biotechnol. 18: 282 - 296.
53 - Reetz, M., Zonta, A., Simplekamp, J. 1996. Efficient immobilization of lipases by
entrapment in hydrophobic sol-gel materials. Biotechnol. Bioeng. 49, 527 – 534.
54 - Kalattukudy, P. 1984. Lipases (Borgström, B. & Brockman, H. Ed.) pp 471-504, Elsevier, Amsterdam, Netherlands.
55 - Lauwereys, M., de Geus , P., Meutter, J., Stanssens, P., Matthyssens, G. 1991. Lipases:
Mechanism and Genetic Engeneering (Alberghina, L., Schimd, R., Verger, R., Eds.) Vol.
16, pp 243-251, VCH, Weinheim, Germany.
56 - Ollis, D., Cheah, M., Dijkstra, B., Frolow, F., Franken, S., Harel, M., Reminghton, S., Silman, J., Schrag, J., Sussnan, K., Verschueren, A. 1992. The α/β hidrolase fold. Protein Eng 5: 197-211
57 - Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G., Cambillau, C. 1992.
Fusarium solani cutinase is a Lipolytic Enzyme with a Catalytic Serine Acessible to
Solvent . Nature. 356; 615-618.
58 - Martinez, C., Nicolas, A., van Tilbeurgh, H., Egloff, M.,Cudry, C., Cambillau, C. 1994.
Cutinase a Lipolitic Enzyme with a Preformed Oxyanion Hole. Biochemistry. 33; 83-89.
59 - Van Tilbeurgh, H., Egloff, M., Martinez, CF., Rugani, N., Verger, R., Cambilau, C. 1993. The Interfacial Activation of the Lipase-procolipase Complex by Mixed Micelles
Revealed by X-ray Crystallography. Nature. 362, 814-820.
60 - Prompers, J., van Noorloos, B., Mannesse, M., Groenewegen, A., Egmond, M., Verheij, 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
entrapment of lipases in hydrophobic sol-gel materials. Angew. Chem. Int. Ed. Engl. 34:
