A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

Abstract

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39-0.40s(-1)) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose

“…A 50 mM sodium acetate buffer, pH 5.0 with 2 mM CaCl 2 , was used throughout unless otherwise stated. Cellobiohydrolase Cel7A from T. reesei and Pyranose Dehydrogenase (PDH) from Agaricus meleagris were heterologously expressed and purified to a single band on an SDS gel and the absence of cellobiase activity in the Cel7A stock in was confirmed as lack of detectable activity against the chromogenic substrate analogue para‐nitrophenyl‐glucopyranoside (results not shown) as described elsewhere (Cruys‐Bagger et al, ; Westh et al, ). Protein concentration was measured at 280 nm in a Nanodrop Spectrophotometer (Thermo Scientific) using a theoretical molar extinction coefficient (Gasteiger et al, ) of 86.7 and 67.8 mM −1 cm −1 for Cel7A and PDH, respectively.…”

“…In the work presented here, we have addressed the issue by implementing a new assay based on a pyranose dehydrogenase (PDH, EC 1.1.99.29) amperometric biosensor (Cruys‐Bagger et al, ). This sensor quantifies soluble sugars in real time over a wide concentration range, allowing direct measurements of the initial, quasi‐steady state rate even in samples with a high background of product (i.e., inhibitor).…”

Exo‐exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo‐lytic character of the enzymes

“…PDH biosensors were prepared according to a previously published protocol (Cruys-Bagger et al, 2014) except that benzoquinone was used as mediator (instead of 2.6-dichlorophenolindophenol). PDH biosensors were prepared according to a previously published protocol (Cruys-Bagger et al, 2014) except that benzoquinone was used as mediator (instead of 2.6-dichlorophenolindophenol).…”

Experimentation and numerical modeling of cyclic voltammetry for electrochemical micro-sized sensors under the influence of electrolyte flow

“…10 Cyclic voltammetry is widely utilized as the main mode of operation for biochemical sensing devices [1,10,11,12,13] and is also useful for probing electroactive surfaces in electrical energy storage and conversion devices [14,15,16].…”