d-Amino acid oxidase bio-functionalized platforms: Toward anenhanced enzymatic bio-activity

ELISA HERRERA; JAVIER VALDEZ TAUBAS; CARLA E. GIACOMELLI

APPLIED SURFACE SCIENCE

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2015 vol. 365 p. 679 - 686

0169-4332

tThe purpose of this work is to study the adsorption process and surface bio-activity of His-taggedd-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the devel-opment of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a)the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanismof His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO−)and presence of Ni(II) (substrate/COO−+ Ni(II)) determined by reflectometry, and (c) the bio-activity ofthe His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorptionbehavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding thecontribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzy-matic performance in solution is highly improved when compared to the previously used pig kidney (pk)DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low asthose expected on a bio-functional platform; hence, it is a viable bio-recognition element of d-amino acidsto be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibita higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modi-fied substrates present enhanced bio-activity because the surface complexes histidine?Ni(II) providewith site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent perfor-mance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinityinteractions whose prevalence depends on the degree of surface coverage.