A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

Amperometric enzymatic sensing of glucose using porous carbon nanotube films soaked with glucose oxidase.

Glucose oxidase was soaked into a porous carbon nanotube film coating on a platinum disk electrode, then trapped beneath a topcoat of electrodeposition paint. The resulting sensors, operated at a potential of +0.6 V (vs. Ag/AgCl), produced a glucose signal that was linear up to 40 mM, with a 50 µM detection limit. Signal stability over >100 h of continuous operation in a flow cell showed the remarkable functional durability of the biosensor, and confirmed that the electropaint coating effectively prevented loss of the enzyme. This performance is deemed to derive from the minimalistic immobilization layer design and the prevention of protein leakage. The immobilization method has a potentially wide scope, in that it may also be applicable in other types of enzymatic biosensor. Graphical abstract Illustration of an enzyme biosensor design that uses glucose oxidase in bare carbon nanotube electrode modifications with electropaint topcoat for amperometric glucose quantification. Immobilization matrix supplementation with extra functional (nano-) materials was unnecessary for high-quality and stable analysis performance.