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

VIALE, FABRIZIO EMILIANO; TAMARA B. BENZAQUÉN; ELIAS, VERÓNICA RITA; GOYA ROSSETTI, GERARDO; EIMER, GRISELDA A.; FERRERO, GABRIEL ORLANDO

Rosario

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

Red Argentina de Tecnología Enzimática

Background: The global trend towards the application of sustainable technologies and the elimination of toxic contaminants has given rise to the use of enzymes as substitutes or complements to traditional catalysts, since they are molecules of incredible selectivity that work at moderate temperatures and pH. In this sense, the use of mesoporous materials modified with transition metals (Cr and Ti) is proposed to generate superoxide radicals and to support the enzymes 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 in situ, which will be used by HRP to oxidize organic matter. On this occasion, the optimization of the final stage of the chemoenzymatic cascade is presented, for which the activity of free HRP and immobilized on SBA-15 or Cr/Ti/SBA-15 supports was evaluated.Methods: SBA-15 mesoporous silicas were synthesized using the sol-gel method, using tetraethylorthosilicate (TEOS) as silicon source, 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 material obtained 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-15 and Cr/Ti/SBA-15. The mesoporous support was suspended in a 0.1 mg/mL enzyme solution of HRP in 25 mM phosphate buffer, pH 7. The degree of enzyme immobilization on the supports was assessed by measuring the absorbance of the immobilization supernatants and the free enzyme solution 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 equipped with a magnetic stirrer at room temperature. The reactors had 10 mg of mesoporous material per reaction or 200 µL of 0.1 mg/ml HRP solution in the case of reactions with free enzyme. The phenol concentrations in each sample were determined by HPLC-UV.Results: The structure of the synthesized SBA-15 material was corroborated by XRD, which was then modified with Cr and Ti metals 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 HRP immobilized on SBA-15 (HRP/SBA-15) and Cr/Ti/SBA-15 (HRP/Cr/Ti/SBA-15) supports was evaluated, working with different initial H2O2 concentrations (0, 0.7, 1.4, 2.8 mM) in 25 mM phosphate buffer, pH 7 with 20 ppm phenol as model contaminant (5 mL reaction volume). The reaction was carried out including a 45 minutes lapse for the adsorption of phenol on the surface of the mesoporous materials in the heterogeneous phase (time 0), taking samples at 30, 60 and 120 minutes. No phenol degradation was detected when using the supports or the supports with the enzyme immobilized in the absence of H2O2. However, HRP activity was optimal at a 1.4 mM H2O2 concentration, with most of the reaction being obtained in the first few minutes, indicating the need for H2O2 as a cofactor. On the other hand, the activity of the HRP/Cr/Ti/SBA-15 biocatalyst was lower than that of HRP/SBA-15, probably due to the lower active surface of the Cr/Ti/SBA-15 material to accommodate phenol molecules. Conclusions: It was possible to immobilize the HRP enzyme on the SBA-15 and Cr/Ti/SBA-15 supports to optimize its activity as a function of the H2O2 concentration managing to degrade 53%