Application of cellulose‐based self‐assembled tri‐enzyme system in a pseudo‐reagent‐less biosensor for biogenic catecholamine detection

Abstract

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.

Laccase immobilization on the tailored cellulose-based Granocel carriers

“…Expected activities are rather low and the per cent of bound activity units (referred to activity units given for immobilization) was ranged from 5.4 to 6.5 in the case of adsorption in buffer and from 7.8 to 12.9 when NaCl was added. Even the lack of affinity of Trametes reesei laccase for cellulose material has been reported recently [26].…”

High‐throughput screening for cellobiose dehydrogenases by Prussian Blue in situ formation

“…We have earlier applied a multienzyme system containing CDH and cellulolytic enzymes absorbed by a cellulosic carrier for a “pseudo‐reagent‐less” reduction of electron acceptors in a biosensor with signal amplification [55]. This system is capable of controllable reduction of ferricyanide with endogenously produced cellobiose in very mild (pH 3.5–4.5) aerobic conditions and the in situ deposition of PB on cellulosic template in the presence of Fe 3+ ions.…”

Predominant Nonproductive Substrate Binding by Fungal Cellobiohydrolase I and Implications for Activity Improvement

“…Absence of saccharifying cellulase activity in CDH has been proven by the lack of dichlorophenol indophenol reduction in the presence of phosphoric acid‐swollen cellulose (filter paper and MCC) as described . Following reported activity of Monilia sp .…”