Explorando técnicas e materiais para a preparação de catalisadores enzimáticos via nanopartículas magnéticas e coimobilização de lipases

Abstract

This communication aimed to explore the techniques and materials used in the immobilization process for enzymatic catalysts development. The experiments were conducted in two stages: in the first stage, Lipase from Rhizomucor miehei (RML) was immobilized by adsorption on magnetite (Fe3O4), magnetic nanoparticles (MNPs), coated with 3-aminopropyltriethoxysilane (APTES), called Fe3O4@APTES-RML or RML-NPM (immobilization yield: 94.7 ± 1.0%; biocatalyst activity: 341.3 ± 1.2 Up-NPB/g), and by covalent bonding activated with glutaraldehyde (GLU) (immobilization yield: 91.9 ± 0.2%; biocatalyst activity: 199 .6 ± 3.5 Up-NPB/g). RML-NPM was characterized by X-ray Powder Diffraction (XRPD), Fourier-Transform Infrared Spectroscopy (FTIR), and Transmission Electron Microscopy (TEM), proving the incorporation of magnetite and the immobilization of RML on the APTES matrix. The results demonstrate that the immobilized biocatalyst exhibited at 60 °C a half-life of 16-19 times superior to the soluble lipase in the pH range 5-10. RML and RML-NPM showed greater activity at pH 7; the immobilized enzyme was more active than the free enzyme in the pH range (5-10) analyzed. Moreover, RML-NPM was applied to the production of ethyl ester from free fatty acids from residual babassu oil, under optimized conditions (30 ºC, 4 h, 1:1 (AGLs/alcohol) determined by the Taguchi method, achieving a conversion of 78.9 ± 0.3% using 9% RML-NPM. Whereas, in the second step, Lipase A from Candida antarctica (CALA) and lipase B from Candida antarctica (CALB) were simultaneous co-immobilization onto chitosan (CHI) and activated with glutaraldehyde, labeled CALA-CALB-CHI, by covalent bonding; this process was optimized using the Taguchi method. Under optimized conditions (pH 9, 5mM, 6: 1 (protein load/g of support) and 1 hour), it was possible to reach 80.00 ± 0.01% for the immobilization yield (IY) and 46.01 ± 0.35 U/g for derivative activity (AtD); in this case, protein charge and ionic strength were the only parameters that have statistical significance and, therefore, those that most influenced the immobilization process. At pH 7, CALA-CALB-CHI showed a half-life of 2-6 times greater than the mixture of CALA and CALB over a temperature range of 50-80 °C. In the pH range between 5 and 9, CALA-CALB showed greater activity at pH 7, while CALA-CALB-CHI, except at pH 7, was more active than the mixture of soluble lipases, especially at pH 9. CHI, CHI-GLU, and CALA-CALB-CHI were characterized by XRPD, FTIR, Scanning Electron Microscopy (SEM), Thermogravimetry (TGA), and Energy Dispersive X-ray Spectroscopy (EDS), proving the immobilization of CALA and CALB on chitosan. Besides, the CALA-CALB-CHI derivative was evaluated in the kinetic resolution of halohydrins rac-2-bromo-1-(2-chlorophenyl) ethyl acetate (2a) and rac-2-chloro-1-(2,4-dichlorophenyl) acetates ethyl acetate (2b), to produce the corresponding halohydrins 3a-b, which are intermediates in the synthesis of the drugs clorprenaline (antiarrhythmic) and luliconazole (antifungal), respectively. (S)-bromohydrin (3a) was obtained with 79% enantiomeric excess (ee), while (S)-chlorohydrin (3b) was produced with 98% ee, 46% conversion, and E>200. Additionally, molecular docking was performed to elucidate the hydrolysis interaction reaction between β-halohydrin acetates and the lipases CALA and CALB.