Building a heterogeneous chemo-enzymatic cascade: optimization of immobilized peroxidase Activity.

FABRIZIO, VIALE; BENZAQUÉN, TAMARA B.; ELÍAS, VERÓNICA R; GOYA ROSSETI, GERARDO FABIAN; EIMER, GRISELDA A.; FERRERO, GABRIEL O.

Rosario

Congreso; Encuentro & Workshop de la Red Argentina de Tecnología Enzimática.; 2023

Red de Argentina de Tecnología Enzimática

The global trend towards the application of sustainable technologies and theelimination of toxic contaminants has given rise to the use of enzymes as substitutes orcomplements to traditional catalysts, since they are molecules of incredible selectivity that work atmoderate temperatures and pH. In this sense, the use of mesoporous materials modified withtransition metals (Cr and Ti) is proposed to generate superoxide radicals and to support theenzymes superoxide dismutase (SOD) and horseradish peroxidase (HRP). In the proposed chemo-enzymatic cascade, the generated superoxides will be a substrate for SOD to generate H2O2 insitu, which will be used by HRP to oxidize organic matter. On this occasion, the optimization of thefinal stage of the chemoenzymatic cascade is presented, for which the activity of free HRP andimmobilized on SBA-15 or Cr/Ti/SBA-15 supports was evaluated.Methods: SBA-15 mesoporoussilicas were synthesized using the sol-gel method, using tetraethylorthosilicate (TEOS) as siliconsource, pluronic acid P123 as structure directing agent, and HCl for hydrolysis and pH adjustment.The resulting solid was filtered, washed, dried and subsequently calcined. Part of the materialobtained was modified post-synthesis by wet impregnation to incorporate Cr (2.5%) and Ti (20%)into the silica matrix.Then, the HRP enzyme was immobilized on the synthesized supports SBA-15and Cr/Ti/SBA-15. The mesoporous support was suspended in a 0.1 mg/mL enzyme solution ofHRP in 25 mM phosphate buffer, pH 7. The degree of enzyme immobilization on the supports wasassessed by measuring the absorbance of the immobilization supernatants and the free enzymesolution by UV/visible spectrophotometry, considering the molar absorptivity coefficient of HRP(102000 M-1cm-1) at 403 nm. The reaction was carried out in 10 mL glass batch reactors equippedwith a magnetic stirrer at room temperature. The reactors had 10 mg of mesoporous material perreaction or 200 µL of 0.1 mg/ml HRP solution in the case of reactions with free enzyme. Thephenol concentrations in each sample were determined by HPLC-UV.Results: The structure of thesynthesized SBA-15 material was corroborated by XRD, which was then modified with Cr and Timetals to obtain the Cr/Ti/SBA-15 support. The HRP enzyme was immobilized on these materials,achieving an 89% immobilization. Subsequently, the activity of free HRP enzyme and HRPimmobilized on SBA-15 (HRP/SBA-15) and Cr/Ti/SBA-15 (HRP/Cr/Ti/SBA-15) supports wasevaluated, working with different initial H2O2 concentrations (0, 0.7, 1.4, 2.8 mM) in 25 mMphosphate buffer, pH 7 with 20 ppm phenol as model contaminant (5 mL reaction volume). Thereaction was carried out including a 45 minutes lapse for the adsorption of phenol on the surfaceof the mesoporous materials in the heterogeneous phase (time 0), taking samples at 30, 60 and120 minutes. No phenol degradation was detected when using the supports or the supports withthe enzyme immobilized in the absence of H2O2. However, HRP activity was optimal at a 1.4 mMH2O2 concentration, with most of the reaction being obtained in the first few minutes, indicatingthe need for H2O2 as a cofactor. On the other hand, the activity of the HRP/Cr/Ti/SBA-15biocatalyst was lower than that of HRP/SBA-15, probably due to the lower active surface of theCr/Ti/SBA-15 material to accommodate phenol molecules. Conclusions: It was possible toimmobilize the HRP enzyme on the SBA-15 and Cr/Ti/SBA-15 supports to optimize its activity as afunction of the H2O2 concentration managing to degrade 53%