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

FRESCURA, ROCÍO; BRUERA, FLORENCIA ALEJANDRA; SADAÑOSKI, MARCELA ALEJANDRA; VELÁZQUEZ, JUAN ERNESTO; KRAMER, GUSTAVO KRAMER; FONSECA, MARÍA ISABEL; ARES, ALICIA ESTHER; ZAPATA, PEDRO DARIO

Rosario

Workshop; V Meeting & II Workshop of the Argentine Network of Enzymatic Technology; 2023

Red Argentina de Tecnología Enzimática

Current biotechnology has revolutionized modern industry through the development and applicationof bionanocatalysts, capable of modifying and even replacing many of the processes based onheterogeneous catalytic reactions. The development of these catalysts consists of theimmobilization of enzymes on supports made of nanostructured materials, thus combining the highselectivity of the catalyst and the mechanical properties of the support. This involves significantimprovements in bionanocatalyst reuse, increased enzyme-substrate contact efficiency, postreaction recovery, and reduced process costs. In this sense, by combining oxidoreductase enzymessuch as Laccase (Lacc), which oxidize a wide range of phenolic compounds in the presence ofmolecular oxygen, and nanostructures of anodic aluminum oxide (AAO) as enzymatic support, it ispossible to develop bionanocatalysts for the treatment of effluents containing recalcitrantpollutants. Since there are different methods of enzymatic immobilization, the objective of this workis to determine the kinetic parameters of Lacc immobilized on AAO nanopores by adsorption andcovalent binding for the development of bionanocatalysts. For this, nanostructured AAO coatingswere synthesized by anodic oxidation from a sheet of AA1050 for 1 h at 30 V, using 0.3 M oxalic acidas electrolyte at 40°C and crude laccase enzyme extract was produced from the culture of thePhlebia brevispora BAFC 633 strain and subsequent precipitation of the supernatant with (NH4)2SO451.6% 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, firstreacting 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 withthe solution of Lacc + buffer. The enzymatic study for the Lacc-AAO bionanocatalysts was performedby measuring the specific activity Laccase (SALacc) in a UV-visible spectrophotometer at 469 nmusing 2,6-dimethoxyphenol as substrate in 0.1 M sodium acetate buffer (pH 3.6) in the range ofconcentrations 0.1-2 mM. The results obtained were adjusted by nonlinear regression using theMichaelis-Menten equation. The Km and Vmax were calculated to be 0.011 mM and 0.23 U cm−2 s−1for the bionanocatalyst synthesized by covalent bonding, and 3.64 mM and 4.14 U cm−2 s−1for thebionanocatalyst synthesized by adsorption, respectively. This demonstrates an improvement in theaffinity and rate of reaction kinetics for the Lacc-AAO bionanocatalysts obtained by adsorption.