Imobilização de lipase de Rhizomucor Miehei em suportes poliméricos e sua aplicação em reações para obtenção de produtos de interesse farmacêutico em sistema contínuo

Abstract

The pharmacological compounds derived from myo-inositol not only present molecules with chiral centers but also hold significant relevance in conditions such as Chagas disease and Parkinson's disease, motivating studies to achieve their purity and enantiomeric resolution. In this context, lipases are excellent catalysts capable of acting in various types of chemical reactions, such as hydrolysis, esterification, and transesterification. To enhance enzymatic stability and activity, immobilization of these enzymes on solid supports through adsorption methods has been employed, which also allows for the reuse of the biocatalyst and its application in continuous systems. In this work, the immobilization and characterization of Rhizomucor Miehei lipase (RML), one of the five most widely used lipases in biotechnology, was carried out on hydrophobic core-shell polymeric supports (CSP) and Accurel (a polypropylene support), as there are currently no reports in the literature combining the use of RML with these support morphologies. The primary objective was to apply the produced biocatalysts in pharmaceutical interest reactions conducted in continuous systems. The CSPs used were P(S-co-DVB)/P(S-co-DVB) and PMMA/PMMA. The evaluated immobilization parameters were immobilization efficiency (%) and immobilization yield (%). The immobilization efficiency of RML on the LRM-P(S-co-DVB)/P(S-co-DVB) support was 71%, with 40 mg of protein/g of support. For the PMMA/PMMA support, the immobilization efficiency was 77.8%, with approximately 40 mg of protein/g of support. The immobilization efficiency for the commercial support Accurel was only 49.5% (with approximately 40 mg of protein/g support). After immobilization, the biocatalyst was characterized through hydrolytic activity assays at different pH (5, 7, and 9) and a range of temperatures (30°C – 60°C). The produced biocatalysts were employed in esterification reactions using oleic acid and ethanol (molar ratio 1:1). LRM-P(S-co-DVB)/P(S-co-DVB) showed esterification activity (16.2 U/g support) superior to LRM-PMMA/PMMA (10.6 U/g support) but inferior to LRM-Accurel biocatalyst (20.7 U/g support). The commercial immobilized version, RM-IM, had esterification activity lower than the biocatalysts produced in this work (2.7 U/g support). The biocatalysts were tested for their catalytic potential in hydrolysis reactions. The highest hydrolytic activity, using p-nitrophenyl laurate as a substrate, was obtained with the LRM-PMMA/PMMA biocatalyst (458.8 U/g support). Considering that lipases are enzymes with high regio-, chemo-, and enantioselectivity, the synthesized biocatalysts were used for the kinetic resolution of (±)-1,3,4-tri-O-benzyl-myo-inositol (rac-1) and (±)-3,6-di-O-benzyl-1,2-O-isopropylidene-myo-inositol (rac-2) derivatives in a continuous system. The product obtained from rac-1 was L-(-)-2-O-acetyl-(±)-1,3,4-tri-O-benzyl-myo-inositol with 36.2% conversion, eep (enantiomeric excess) >99, and productivity of 0.059 mg product/[mg enzyme x h], and the product from rac-2 was L-5-O-acetyl-3,6-di-O-benzyl-1,2-O-isopropylidene-myo-inositol, with 48.2% conversion, eep >99, and productivity of 0.052 mg product/[mg enzyme x h]. The reuse of the biocatalyst was evaluated over 10 consecutive cycles without significant reduction in conversion, obtaining promising results for the kinetic resolution of myo-inositol derivatives.