Study of the enzymatic kinetics of laccase immobilized on nanoporous aluminum oxide for the development of bionanocatalysts

FRESCURA R.V.; BRUERA F.A.; SADAÑOSKI M. A.; VELÁZQUEZ J.E.; KRAMER G.R.; FONSECA M.I.; ARES A.E.; ZAPATA P.D.

Rosario

Workshop; V ENCUENTRO & II WORKSHOP DE LA RED ARGENTINA DE TECNOLOGÍA ENZIMÁTICA; 2023

Red Argentina de Tecnología Enzimática

Current biotechnology has revolutionized modern industry through the development and application of bionanocatalysts, capable of modifying and even replacing many of the processes based on heterogeneous catalytic reactions. The development of these catalysts consists of the immobilization of enzymes on supports made of nanostructured materials, thus combining the high selectivity of the catalyst and the mechanical properties of the support. This involves significant improvements in bionanocatalyst reuse, increased enzyme-substrate contact efficiency, post-reaction recovery, and reduced process costs. In this sense, by combining oxidoreductase enzymes such as Laccase (Lacc), which oxidize a wide range of phenolic compounds in the presence of molecular oxygen, and nanostructures of anodic aluminum oxide (AAO) as enzymatic support, it is possible to develop bionanocatalysts for the treatment of effluents containing recalcitrant pollutants. Since there are different methods of enzymatic immobilization, the objective of this work is to determine the kinetic parameters of Lacc immobilized on AAO nanopores by adsorption and covalent binding for the development of bionanocatalysts. For this, nanostructured AAO coatings were synthesized by anodic oxidation from a sheet of AA1050 for 1 h at 30 V, using 0.3 M oxalic acid as electrolyte at 40°C and crude laccase enzyme extract was produced from the culture of the Phlebia brevispora BAFC 633 strain and subsequent precipitation of the supernatant with (NH4)2SO4 51.6% m v-1. Next, Lacc was immobilized on the AAO coatings for 1 h using a solution of Lacc + sodium acetate buffer (pH = 4.4) with a final concentration of 575 U L-1, by: a) physical adsorption, immersing the supports in a solution of Lacc + buffer with stirring and b) covalent binding, first reacting the support with 3-amino-propyltriethoxysilane and triethanolamine in dry acetonitrile (ACN), then with N,N´-carbonyldiimidazole in ACN and finally immersing the activated supports with the solution of Lacc + buffer. The enzymatic study for the Lacc-AAO bionanocatalysts was performed by measuring the specific activity Laccase (SALacc) in a UV-visible spectrophotometer at 469 nm using 2,6-dimethoxyphenol as substrate in 0.1 M sodium acetate buffer (pH 3.6) in the range of concentrations 0.1-2 mM. The results obtained were adjusted by nonlinear regression using the Michaelis-Menten equation. The Km and Vmax were calculated to be 0.011 mM and 0.23 U cm−2 s−1 for the bionanocatalyst synthesized by covalent bonding, and 3.64 mM and 4.14 U cm−2 s−1 for the bionanocatalyst synthesized by adsorption, respectively. This demonstrates an improvement in the affinity and rate of reaction kinetics for the Lacc-AAO bionanocatalysts obtained by adsorption.