From Graphite to Laccase Biofunctionalized Few-Layer Graphene: A “One Pot” Approach Using a Chimeric Enzyme

Abstract

A chimeric enzyme based on the genetic fusion of a laccase with a hydrophobin domain was employed to functionalize few-layer graphene, previously exfoliated from graphite in the presence of the hydrophobin. The as-produced, biofunctionalized few-layer graphene was characterized by electrochemistry and Raman spectroscopy, and finally employed in the biosensing of phenols such as catechol and dopamine. This strategy paves the way for the functionalization of nanomaterials by hydrophobin domains of chimeric enzymes and their use in a variety of electrochemical applications.

Innovative surface bio-functionalization by fungal hydrophobins and their engineered variants

“…In this case, an added value of the system was the enzyme immobilization on carbon nanomaterials, thereby endowed with a high surface-to-volume ratio. Starting from previous experimental works that demonstrated the successful functionalization of nanomaterials with Vmh2 (Gravagnuolo et al, 2015), Sorrentino et al (2020b) produced Frontiers in Molecular Biosciences frontiersin.org few-layer graphene functionalized with the Vmh2-laccase fusion protein through a "one-pot" approach. Once the immobilization conditions were optimized, biofunctionalized few-layer graphene was deposited on glass carbon electrodes to detect catechol at micromolar sensitivity.…”

Laccase-catalyzed functionalization of phenol-modified carbon nanotubes: from grafting of metallopolyphenols to enzyme self-immobilization

“…Laccases have also been employed in electrochemical applications, mostly biosensors and biofuel cells, relying on the efficient immobilization and electrical wiring of laccases at the electrode surface. 3,4,[11][12][13][14][15] LAC3 is a typical fungal laccase which can be obtained with high yield as recombinant protein in yeast. 16,17 We have already exploited its excellent versatility and performances in several applications.…”

The laccase mediator system at carbon nanotubes for anthracene oxidation and femtomolar electrochemical biosensing

“…[16][17][18][19] We recently produced in Pichia pastoris a chimera by fusion of POXA1b with a hydrophobin, a self-assembling adhesive protein produced by the same fungus P. ostreatus, making laccase prone to be immobilized at different types of materials such polystyrene beads, graphene nanosheets or CNTs. 16,18,20 Laccase redox mediators, the most common being the diammonium salt of 2,2′-azine-bis(3-ethylbenzothiazoline-6sulfonic acid (ABTS), have been used for many years, acting as electron relays when laccase are immobilized at electrode surface 12,13 or acting as an enhancer/promoter of the ability of laccase to catalyze the oxidation of high potential nonphenolic substrate. 21 Several studies have shown that this so-called laccase-mediator system is able to oxidize several PAHs such as anthracene or benzo(a)pyrene into quinoid products.…”

“…12 For biosensing applications, laccases have been mostly studied for the detection of ortho -diphenol substrates such as catechol or dopamine. 16–19 We recently produced in Pichia pastoris a chimera by fusion of POXA1b with a hydrophobin, a self-assembling adhesive protein produced by the same fungus P. ostreatus , making laccase prone to be immobilized at different types of materials such polystyrene beads, graphene nanosheets or CNTs. 16,18,20…”