2004-01-14 18:17:00WuYung123
蛋白質工程用於生物電子催化 (1)
Protein engineering in bioelectrocatalysis: (蛋白質工程用於生物電子催化)
(based on Wong and Schwaneberg (2003))
Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells (e.g. for powering miniaturised implanted medical devices) and chemical syntheses of pharmaceuticals and fine chemicals. Central to this approach is the efficiency of electron transfer between electrode surfaces and redox proteins. Research in interface design is shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering methods, like rational design, directed evolution and combination of both are now typical tools used to aid in improving electron transfer properties of redox proteins.
Electron transfer plays a important role in essential life activities like photosynthesis, respiration and metabolic pathways. Redox proteins account for approximately one-quarter of all known proteins, and these proteins shuttles electrons and catalysing oxidation and reduction reaction. Natural selection prevents random electron transfer in living organisms and ensures that valuable energy resources are not wasted and that toxic metabolites such as hydrogen peroxide are not produced. Three factors determine electron transfer within proteins:
1. Reorganisation energies-qualitatively reflecting the structural rigidity in the oxidised and reduced form
2. Potential differences and orientations of involved redox sites
3. Distances between redox active sites and the intervening medium
Bioelectrocatalysis still in its infancy have to faced with a lot of challenges, and protein engineering can improve enzymatic properties and enhances electrochemical performance of bioelectronics through trimming the protein, protein surface modification, active site mutations and domain shuffling, thereby overcoming the following challenges:
1. Field conditions: proteins activities are strongly dependent on the working environment e.g. pH, salt conc. and ionic strength.
2. Enzyme properties:
a. Cofactor requirement
Cofactor regeneration is a major challenge for many redox protein applications, as cofactors like NADPH or NADH are expensive and tend to adsorbed at electrode surfaces, and subsequently be oxidised, which can even modified the electrode surface.
b. Enzyme instability
Maximum signal output can not be maintained if protein is degrade in a short period of time and irreversible modify electrode surface. In the case of aerobic cathodic systems, reduction of oxygen generates superoxide or peroxide like reactive species that can damage the biocatalyst.
c. Enzyme pre-treatment
Oxidative pretreatment for cleaving the sugar moieties of redox proteins in eurkaryotics can speed up electron transfer rates by reducing the distance between electron donor and acceptor pair. However strong oxidising agents can often reduce enzyme activity.
d. Protein immobilisation
Protein immobilisation onto electrode surfaces to achieve reproducible and fast electron transfer is often a trial and error process. Technical challenges include the even distribution of enzymes on electrode surfaces, and the modification of enzymes or electrode surfaces to provide linkages and reduction of enzyme activity due to immobilisation.
e. Electron transfer mechanism
Regulation of electron transfer is highly advanced and well gated, e.g. for P450 BM-3, in the absence of substrate, no electron transfer from FMN to hame occurs, thereby preventing consumption of energy resources and avoiding the generation of reactive species.
3. Interface
Heterogeneous et rates is one of the limiting factor of the performance and efficiency of bioelectrochemical devices. Progress were made in electrode design, surface functionalisation for protein immobilisation, irreversible adsorption and the degradation of cofactors… etc, by going though the different routes like mediated electron transfer, direct electron transfer between proteins and electrodes, electron transfer cascades via redox hydrogels, and conducting polymer films.
(based on Wong and Schwaneberg (2003))
Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells (e.g. for powering miniaturised implanted medical devices) and chemical syntheses of pharmaceuticals and fine chemicals. Central to this approach is the efficiency of electron transfer between electrode surfaces and redox proteins. Research in interface design is shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering methods, like rational design, directed evolution and combination of both are now typical tools used to aid in improving electron transfer properties of redox proteins.
Electron transfer plays a important role in essential life activities like photosynthesis, respiration and metabolic pathways. Redox proteins account for approximately one-quarter of all known proteins, and these proteins shuttles electrons and catalysing oxidation and reduction reaction. Natural selection prevents random electron transfer in living organisms and ensures that valuable energy resources are not wasted and that toxic metabolites such as hydrogen peroxide are not produced. Three factors determine electron transfer within proteins:
1. Reorganisation energies-qualitatively reflecting the structural rigidity in the oxidised and reduced form
2. Potential differences and orientations of involved redox sites
3. Distances between redox active sites and the intervening medium
Bioelectrocatalysis still in its infancy have to faced with a lot of challenges, and protein engineering can improve enzymatic properties and enhances electrochemical performance of bioelectronics through trimming the protein, protein surface modification, active site mutations and domain shuffling, thereby overcoming the following challenges:
1. Field conditions: proteins activities are strongly dependent on the working environment e.g. pH, salt conc. and ionic strength.
2. Enzyme properties:
a. Cofactor requirement
Cofactor regeneration is a major challenge for many redox protein applications, as cofactors like NADPH or NADH are expensive and tend to adsorbed at electrode surfaces, and subsequently be oxidised, which can even modified the electrode surface.
b. Enzyme instability
Maximum signal output can not be maintained if protein is degrade in a short period of time and irreversible modify electrode surface. In the case of aerobic cathodic systems, reduction of oxygen generates superoxide or peroxide like reactive species that can damage the biocatalyst.
c. Enzyme pre-treatment
Oxidative pretreatment for cleaving the sugar moieties of redox proteins in eurkaryotics can speed up electron transfer rates by reducing the distance between electron donor and acceptor pair. However strong oxidising agents can often reduce enzyme activity.
d. Protein immobilisation
Protein immobilisation onto electrode surfaces to achieve reproducible and fast electron transfer is often a trial and error process. Technical challenges include the even distribution of enzymes on electrode surfaces, and the modification of enzymes or electrode surfaces to provide linkages and reduction of enzyme activity due to immobilisation.
e. Electron transfer mechanism
Regulation of electron transfer is highly advanced and well gated, e.g. for P450 BM-3, in the absence of substrate, no electron transfer from FMN to hame occurs, thereby preventing consumption of energy resources and avoiding the generation of reactive species.
3. Interface
Heterogeneous et rates is one of the limiting factor of the performance and efficiency of bioelectrochemical devices. Progress were made in electrode design, surface functionalisation for protein immobilisation, irreversible adsorption and the degradation of cofactors… etc, by going though the different routes like mediated electron transfer, direct electron transfer between proteins and electrodes, electron transfer cascades via redox hydrogels, and conducting polymer films.