Novas fronteiras em biocatálise lipases imobilizadas em quitosana-EDTA com aplicação na resolução de (R,S)-1-feniletanol e lipases livres em reações sequenciais e multicomponentes

Abstract

Chemistry is constantly advancing toward environmental compatibility, making Green Chemistry the ultimate goal of most academic and industrial projects. In this context, the application of enzymes as catalysts for chemical reactions has gained prominence. Enzyme immobilization, in turn, addresses issues related to the solubility and stability of free enzymes. Furthermore, the development of techniques and the search for new supports improve other

properties, such as activity, selectivity, inhibition, and chemical resistance. Chitosan is a well- known support in enzymatic technology, with one of its main features being its ability to

undergo chemical modifications. This study focused on the functionalization of chitosan with EDTA (CHT-EDTA) and its application as a support for the immobilization of lipases from Burkholderia cepacia (BCL), Candida rugosa (CRL), and Aspergillus niger (ANL). The enzymatic activity and stability of the lipases, before and after conjugation to the support, were evaluated under different conditions (pH, temperature, and organic solvents) and

compared to those of free lipase by monitoring the hydrolysis of p-nitrophenyl palmitate (p- NPP). Conformational changes were investigated using Fourier Transform Infrared

Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Thermogravimetric Analysis (TGA). The adsorption capacity of lipases on the CHT-EDTA surface increased with contact time at an optimal pH value (pH 6.5). The results revealed that 112 mg/g for BCL/CHT-EDTA was the highest amount of adsorbed lipase, followed by CRL with 106 mg/g and ANL with 53 mg/g, corresponding to maximum immobilization efficiencies of 72%, 69%, and 45%, respectively. The immobilized enzymes demonstrated enzymatic

activities of 1,539 U/g for BCL/CHT-EDTA, followed by CRL/CHT-EDTA and ANL/CHT- EDTA with 1,409 and 1,063 U/g, respectively. BCL/CHT-EDTA was then chosen for

studying the resolution of R,S-1-phenylethanol, achieving 99.9% of eeP, 82% of eeS , and 45% conversion (E > 200). BCL/CHT-EDTA maintained high enantioselectivity (eeP, > 99.9%) even after four reuse cycles. The free enzymes were also explored as a strategy for the synthesis of organic molecules, producing compounds under mild and efficient conditions. In this regard, BCL, CRL, ANL, Candida antarctica Lipase A (CALA) immobilized on Immobead 150, and Candida antarctica Lipase B (CALB) immobilized on an acrylic resin were applied as biocatalysts in the synthesis of xanthene, chromene, and spirooxindole derivatives. CRL stood out with a catalytic rate 10 times faster than the other lipases, making it the selected biocatalyst for subsequent reactions. The synthesis of xanthene derivatives (1a-h) was carried out with various salicylaldehydes and dimedone, achieving short reaction times (10–80 min) and high yields (88–94.4%), with CRL recovery and reuse for up to four consecutive cycles. The optimized methodology for xanthenes was also applied to the synthesis of chromene (2a-o) and spirooxindole derivatives (3a-h), employing different benzaldehydes/isatins, dimedone, and malononitrile. Compounds 1 and 2 were evaluated for antimicrobial activity through in vitro assays. Compounds 1d (with -NO2 substituent), 2c (with -Br substituent), and 2e (with -OCH3 substituent) showed promising results, with Minimum Inhibitory Concentrations (MIC) of 128, 64, and 64 μg/mL, respectively, for most of the tested microorganisms.