Optimizing Enzyme Immobilization in Mesoporous Silica - A Spectroscopic Study of the Dynamics and Spatial Distribution of the Confined Enzymes
Doctoral thesis, 2018
Enzymes as biological catalysts are immobilized in porous materials to improve the enzyme activity and simplify their purification from the reaction media in biocatalytic applications. Mesoporous silica (MPS) particles are used as a promising support material for the immobilization of enzymes. The focus of this thesis is placed on the dynamics and distribution of enzymes in confining environments, to gain further understanding of the immobilization mechanism. The work is mainly based on various spectroscopy techniques using enzyme-attached fluorescent probes.
To elucidate the mechanistic steps of the immobilization process, high time resolutions methods are essential. In this thesis, a fluorescence spectroscopy assay is developed to monitor the translational dynamics of the enzymes in real time while immobilization occurs. It is shown that the size of enzyme for a given pore size can strongly affect the kinetics of enzyme immobilization. The rotational dynamics of the confined enzymes is studied using fluorescence anisotropy and it is shown that the rotation of the enzymes is slower inside the pores compared to the enzymes in free solution. In order to investigate if protein-protein or protein-wall hydrodynamic interactions have an impact on retarding the dynamics of the confined enzymes, the microenvironment inside the MPS particles needs to be studied. In this thesis, the microviscosity inside the MPS particles is measured using enzyme-attached carbocyanine dyes. The results show that the effective microviscosity is about ten times higher inside the MPS particles than in bulk water. The increase is stronger with smaller pores and higher enzyme concentration, and it is concluded that protein-wall hydrodynamic interactions probably have a more significant effect in retarding the confined enzymes.
The distribution of two co-immobilized enzymes in a cascade reaction which converts carbon dioxide (CO2) to formaldehyde (CH₂O) is studied. The effect of the distance between the enzymes on the catalytic efficiency is investigated using Förster resonance energy transfer spectroscopy. The results demonstrate that the two immobilized enzymes are in close enough proximity resulting in substrate channeling between the active sites and four times more efficient conversion of CO2 to CH₂O. Finally the location of the immobilized enzymes is visualized using transmission electron microscopy and immunogold staining. Two types of MPS particles are used with different pore morphology, spherical and hexagonal. The results show that not only the size of the pores in the MPS particles is an important factor, but the morphology of the pores also plays a crucial role in optimizing the enzyme immobilization.
To elucidate the mechanistic steps of the immobilization process, high time resolutions methods are essential. In this thesis, a fluorescence spectroscopy assay is developed to monitor the translational dynamics of the enzymes in real time while immobilization occurs. It is shown that the size of enzyme for a given pore size can strongly affect the kinetics of enzyme immobilization. The rotational dynamics of the confined enzymes is studied using fluorescence anisotropy and it is shown that the rotation of the enzymes is slower inside the pores compared to the enzymes in free solution. In order to investigate if protein-protein or protein-wall hydrodynamic interactions have an impact on retarding the dynamics of the confined enzymes, the microenvironment inside the MPS particles needs to be studied. In this thesis, the microviscosity inside the MPS particles is measured using enzyme-attached carbocyanine dyes. The results show that the effective microviscosity is about ten times higher inside the MPS particles than in bulk water. The increase is stronger with smaller pores and higher enzyme concentration, and it is concluded that protein-wall hydrodynamic interactions probably have a more significant effect in retarding the confined enzymes.
The distribution of two co-immobilized enzymes in a cascade reaction which converts carbon dioxide (CO2) to formaldehyde (CH₂O) is studied. The effect of the distance between the enzymes on the catalytic efficiency is investigated using Förster resonance energy transfer spectroscopy. The results demonstrate that the two immobilized enzymes are in close enough proximity resulting in substrate channeling between the active sites and four times more efficient conversion of CO2 to CH₂O. Finally the location of the immobilized enzymes is visualized using transmission electron microscopy and immunogold staining. Two types of MPS particles are used with different pore morphology, spherical and hexagonal. The results show that not only the size of the pores in the MPS particles is an important factor, but the morphology of the pores also plays a crucial role in optimizing the enzyme immobilization.
fluorescence spectroscopy
dynamics
mesoporous silica
Immunogold staining
Transmission electron microscopy
anisotropy
microviscosity
real-time monitoring
Cy5.
Förster resonance energy transfer
Enzyme immobilization
Cy3
KA-Salen, Kemigården 4, Chalmers.
Opponent: Prof. Michelle Digman, University of California, Irvine, USA
Author
Pegah Sadat Nabavi Zadeh
Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry
A fluorescence spectroscopy assay for real-time monitoring of enzyme immobilization into mesoporous silica particles
Analytical Biochemistry,;Vol. 476(2015)p. 51-58
Journal article
Immobilization of Enzymes in Mesoporous Silica Particles: Protein Concentration and Rotational Mobility in the Pores
Journal of Physical Chemistry B,;Vol. 121(2017)p. 2575-2583
Journal article
Measuring Viscosity inside Mesoporous Silica Using Protein-Bound Molecular Rotor Probe, Pegah S. Nabavi Zadeh*, Milene Zezzi do Valle Gomes, Maria Abrahamsson, Anders E.C. Palmqvist, Björn Åkerman Submitted to Physical Chemistry Chemical Physics journal, RSC, 14-Feb-2018
Förster resonance energy transfer study of the improved biocatalytic conversion of CO2 to formaldehyde by co-immobilization of enzymes in siliceous mesostructured cellular foams Pegah S. Nabavi Zadeh#*, Milene Zezzi do Valle Gomes#, Björn Åkerman, Anders E.C. Palmqvist Submitted to ACS Catalysis journal, 16-Feb-2018, under revision. #: Equally contributed as first authors Manuscript
The spatial distribution of enzyme immobilized in mesoporous silica of SBA-15 and MCF-types visualized by TEM and immunogold staining. Pegah S. Nabavi Zadeh*, Milene Zezzi do Valle Gomes, Anders E.C. Palmqvist, Björn Åkerman, Manuscript, January 2018
Enzymes as biological catalysts can accelerate and regulate metabolic reactions in all living cells with high selectivity. Without biocatalysts, the chemical reactions in life process, such as digestion of food and synthesis of DNA could last forever. These days enzymes are used in the food, agricultural, cosmetic, and pharmaceutical industries as an environmentally-friendly alternative to conventional synthetic catalysts. However, the use of enzymes is rather limited due to a low long-term operational stability and difficulties in recycling enzymes from the reaction media. In order to increase the stability of enzymes and reuse them, it is therefore advantageous to immobilize them into solid materials. These solid materials as a support can form a protective environment and the immobilized enzyme can be easily separated from the reaction media.
In my PhD research, mesoporous silica particles were used as a promising support material for immobilization of enzymes. The porous structure provides a protective microenvironment in which the enzymes can tolerate extreme pH and high temperature. These silica particles have a large surface area and pore volume which make them suitable for confining the enzymes. The activity of enzymes after immobilization does not always increase, it is possible even to be reduced. Therefore, in order to use the immobilized enzymes in biocatalytic applications for industries, the immobilization process needs to be optimized based on the support material and the type of enzyme. For this optimization, there is a need to further understand the microenvironment inside the pores and the interactions between the support and enzymes.
In the first part of my thesis, I studied the dynamics of the enzyme immobilization in mesoporous silica particles by developing a fluorescence method and monitoring the process in real-time. Also the microenvironment inside the mesoporous particles was characterized. The effect of changes in microviscosity inside the pores on the dynamics behavior of the confined enzymes was discussed.
In the second part of this thesis, the distribution and location of the immobilized enzymes were studied with spectroscopy and microscopy techniques in different mesoporous silica particles. The effect of the pore morphology on the enzyme loading was studied in order to optimize the immobilization by choosing a suitable mesoporous silica particles. The research presented here can be used to rationally design the enzyme immobilization process for biocatalytic applications and making this method a suitable choice for industries.
In my PhD research, mesoporous silica particles were used as a promising support material for immobilization of enzymes. The porous structure provides a protective microenvironment in which the enzymes can tolerate extreme pH and high temperature. These silica particles have a large surface area and pore volume which make them suitable for confining the enzymes. The activity of enzymes after immobilization does not always increase, it is possible even to be reduced. Therefore, in order to use the immobilized enzymes in biocatalytic applications for industries, the immobilization process needs to be optimized based on the support material and the type of enzyme. For this optimization, there is a need to further understand the microenvironment inside the pores and the interactions between the support and enzymes.
In the first part of my thesis, I studied the dynamics of the enzyme immobilization in mesoporous silica particles by developing a fluorescence method and monitoring the process in real-time. Also the microenvironment inside the mesoporous particles was characterized. The effect of changes in microviscosity inside the pores on the dynamics behavior of the confined enzymes was discussed.
In the second part of this thesis, the distribution and location of the immobilized enzymes were studied with spectroscopy and microscopy techniques in different mesoporous silica particles. The effect of the pore morphology on the enzyme loading was studied in order to optimize the immobilization by choosing a suitable mesoporous silica particles. The research presented here can be used to rationally design the enzyme immobilization process for biocatalytic applications and making this method a suitable choice for industries.
Areas of Advance
Nanoscience and Nanotechnology (SO 2010-2017, EI 2018-)
Life Science Engineering (2010-2018)
Materials Science
Subject Categories
Biochemistry and Molecular Biology
Analytical Chemistry
Biological Sciences
Biocatalysis and Enzyme Technology
ISBN
978-91-7597-718-8
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4399
Publisher
Chalmers
KA-Salen, Kemigården 4, Chalmers.
Opponent: Prof. Michelle Digman, University of California, Irvine, USA