Engineering of Cellobiose Dehydrogenases for Improved Glucose Sensitivity and Reduced Maltose Affinity

Abstract

Cellobiose dehydrogenase (CDH) is a fungal extracellular flavocytochrome capable of direct electron transfer (DET). Unlike other CDHs, the pH optimum for CDHs from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) is close to the human physiological pH in blood (7.4). These are, therefore, interesting candidates for glucose measurements in human blood and the application in enzymatic fuel cells is, however, limited by their relatively low activity with this substrate. In this work, the substrate specificities of CtCDH and HiCDH have been altered by a single cysteine to tyrosine substitution in the active sites of CtCDH (position 291) and HiCDH (position 285), which resulted in improved kinetic constants with glucose while decreasing the activity with several disaccharides, including maltose. The DET properties of the generated CDH variants were tested in the absence and in the presence of substrates, on graphite electrodes and thiolic self‐assembled monolayer (SAM)‐modified Au electrodes. Seven different thiols with different spacer lengths were used, containing ‐COOH, ‐OH, and ‐NH2 end groups. The length and head functionality of the thiol govern the efficiency of the DET reaction and indicate different DET properties of CtCDH and HiCDH

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

“…Especially, DET-type bioelectrocatalysis, in which the electrode and enzymatic reactions are directly coupled, plays an significant role in the construction of mediatorfree and simple bioelectrochemical-devices for biosensing (biosensors) and biochemical electricity production (biofuel cells) with minimum overpotential in theory. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] In order to improve or investigate the DET-type bioelectrocatalysis, several protein-engineering methods have been examined: the point mutation around the active site to change its catalytic characteristics, [17][18][19][20] the deglycosylation to shorten the distance between an electrode and the redox center buried in the enzyme and improve the interfacial electron transfer kinetics, 18,21,22 and the insertion of a tag sequence or cysteine residue(s) to control the orientation of the redox enzyme on gold (Au) electrodes. [23][24][25] D-Fructose dehydrogenase from Gluconobacter japonicus NBRC3260 (FDH; EC 1.1.99.11) is a heterotrimeric membrane protein consisting of subunits I (67 kDa), II (51 kDa), and III (20 kDa).…”

A Third Generation Glucose Biosensor Based on Cellobiose Dehydrogenase Immobilized on a Glassy Carbon Electrode Decorated with Electrodeposited Gold Nanoparticles: Characterization and Application in Human Saliva

“…DH CDH domain is structurally similar to the FAD domain of most GMC-oxidoreductase enzymes and is fully reduced by di-/mono- saccharides, transferring the electrons through internal electron transfer (IET) to the CYT CDH , which finally shuttles the electrons to properly modified electrodes [ 31 ]. Among II class CDHs, Corynascus thermophilus CDH ( Ct CDH) was genetically mutated in its active site ( Ct CDH C291Y mutant) to enhance its sensitivity toward glucose and reduce the maltose cross-reactivity [ 32 ].…”

Detection of cellular metabolites by redox enzymatic cascades

“…For example, engineered enzymes with more enhanced properties of electron transfer can be immobilized in electrodes for use as biosensors. [17,18] Different types of enzymes are suitable for giving different types of ). [20] Bioluminescence catalyzed by luciferases typically gives high reading sensitivity (Figure 1, panel c) and light generated can be measured by a simple and portable photomultiplier equipped luminometer or observed directly by the naked eye or quantified by smartphones.…”