94
5 Protein-Engineering Approach for Improvement …
special properties, such as thermal stability, pH activity/stability, tolerance towards
salts etc [16, 17]. In the view of DET-type bioelectrocatalysis, several enzyme modification approaches have been proposed: (i) trimming of the N- or C-terminals and
deglycosylation to reduce the size of enzymes and to open up the redox active site
[18], (ii) site-directed mutation at the redox active site to change its redox and catalytic
characteristics [19], and (iii) insertion of tag sequences and site-specific mutation of
amino acid residue to control the orientation of enzymes on electrodes [20].
5.2 Enzyme Trimming
Enzymes are biomacromolecules that have usually large and sophisticated threedimensional molecular structures. An effective strategy to increase the interfacial
electron transfer kinetics for DET-type bioelectrocatalysis is downsizing the dimension of enzymes to shorten the distance between the active sites and the electrode
surfaces and to increase the surface concentration of enzymes.
Generally, carbohydrate of enzymes acts as an insulating shell and increases the
distance between the redox active site of enzymes and the electrode surface. Therefore, a useful method to shorten the distance is deglycosylation. For an example,
in previous researches, it was shown that deglycosylation of HRP [21] and tobacco
peroxidase [22] shortened the distance between the prosthetic group and the electrode
surface, thereby enhanced the DET kinetics. HRP is a ferric enzyme that catalyses the
oxidation of various kinds of electron donating substrates with H 2 O 2 as the electron
acceptor. The carbohydrate content of HRP reaches 18 % of the total mass, which
was considered to hinder the electron transfer between the active site and electrodes.
Gorton et al. constructed carbohydrate-free recombinant HRP enzymes [21]. The
engineered enzyme-modified polycrystalline gold electrodes exhibited high current
responses of DET-type bioelectrocatalysis to H 2 O 2, while a native glycosylated HRPmodified electrode didn’t show such catalytic currents. The improvement in the DET
kinetics of the enzymes was ascribed to the absences of the carbohydrate shell on
the recombinant HRPs.
Except to HRP, improved DET-type bioelectrocatalysis was also reported in
other deglycosylated enzymes: cellobiose dehydrogenases (CDH) [23], pyranose
dehydrogenase (PDH) [24], and glucose oxidase (GOD) [25]. GOD is a flavoenzyme
catalyzing glucose oxidation and is widely utilized in glucose biosensors and biofuel
cells. GOD from Aspergillus niger is a heavily glycosylated homodimer that contains
one FAD per monomer as the cofactor (Fig. 5.1A). The molecular mass is ca. 160 kDa
depending on the level of the glycosylation. The calculated value of the minimum
distance between the FAD to the outside of the protein in GOD is ca. 1.7 nm, which
may lead to sluggish direct electron transfer of GOD adsorbed at a solid electrode
[26, 27]. Mano and his co-workers reported a nearly fully deglycosylated and
highly purified GOD that exhibited clear DET response at a glassy carbon electrode
(Fig. 5.1B) [25]. The onset potential of the catalytic glucose oxidation wave obtained
at the deglycosylated GOx-adsorbed electrode was −0.49 V versus Ag|AgCl. The
5 Protein-Engineering Approach for Improvement …
special properties, such as thermal stability, pH activity/stability, tolerance towards
salts etc [16, 17]. In the view of DET-type bioelectrocatalysis, several enzyme modification approaches have been proposed: (i) trimming of the N- or C-terminals and
deglycosylation to reduce the size of enzymes and to open up the redox active site
[18], (ii) site-directed mutation at the redox active site to change its redox and catalytic
characteristics [19], and (iii) insertion of tag sequences and site-specific mutation of
amino acid residue to control the orientation of enzymes on electrodes [20].
5.2 Enzyme Trimming
Enzymes are biomacromolecules that have usually large and sophisticated threedimensional molecular structures. An effective strategy to increase the interfacial
electron transfer kinetics for DET-type bioelectrocatalysis is downsizing the dimension of enzymes to shorten the distance between the active sites and the electrode
surfaces and to increase the surface concentration of enzymes.
Generally, carbohydrate of enzymes acts as an insulating shell and increases the
distance between the redox active site of enzymes and the electrode surface. Therefore, a useful method to shorten the distance is deglycosylation. For an example,
in previous researches, it was shown that deglycosylation of HRP [21] and tobacco
peroxidase [22] shortened the distance between the prosthetic group and the electrode
surface, thereby enhanced the DET kinetics. HRP is a ferric enzyme that catalyses the
oxidation of various kinds of electron donating substrates with H 2 O 2 as the electron
acceptor. The carbohydrate content of HRP reaches 18 % of the total mass, which
was considered to hinder the electron transfer between the active site and electrodes.
Gorton et al. constructed carbohydrate-free recombinant HRP enzymes [21]. The
engineered enzyme-modified polycrystalline gold electrodes exhibited high current
responses of DET-type bioelectrocatalysis to H 2 O 2, while a native glycosylated HRPmodified electrode didn’t show such catalytic currents. The improvement in the DET
kinetics of the enzymes was ascribed to the absences of the carbohydrate shell on
the recombinant HRPs.
Except to HRP, improved DET-type bioelectrocatalysis was also reported in
other deglycosylated enzymes: cellobiose dehydrogenases (CDH) [23], pyranose
dehydrogenase (PDH) [24], and glucose oxidase (GOD) [25]. GOD is a flavoenzyme
catalyzing glucose oxidation and is widely utilized in glucose biosensors and biofuel
cells. GOD from Aspergillus niger is a heavily glycosylated homodimer that contains
one FAD per monomer as the cofactor (Fig. 5.1A). The molecular mass is ca. 160 kDa
depending on the level of the glycosylation. The calculated value of the minimum
distance between the FAD to the outside of the protein in GOD is ca. 1.7 nm, which
may lead to sluggish direct electron transfer of GOD adsorbed at a solid electrode
[26, 27]. Mano and his co-workers reported a nearly fully deglycosylated and
highly purified GOD that exhibited clear DET response at a glassy carbon electrode
(Fig. 5.1B) [25]. The onset potential of the catalytic glucose oxidation wave obtained
at the deglycosylated GOx-adsorbed electrode was −0.49 V versus Ag|AgCl. The