Imobilização da lipase de Pichia pastoris em óxido de grafeno: Uma abordagem integrada a hidrólise enzimática com separação simultânea empregando biorreator de membrana

Abstract

Enzyme technology has experienced notable advancement in the field of biochemistry, highlighting enzymes as highly efficient biological catalysts to drive chemical reactions. At the same time, membrane separation technology has aroused considerable interest, due to its advantages over traditional separation methods. The study proposes the use of graphene oxide as a support for the immobilization of lipase from Pichia pastoris, using the immobilized and free biocatalyst, integrated into the investigation of the production of cellulose acetate membranes applied in enzymatic hydrolysis with simultaneous separation of residual soybean oil. The best enzyme loading, optimum temperature and pH and immobilization time were defined, as well as thermal stability and d eactivation Kinetic and Thermodynamic parameters were defined for the free and immobilized enzyme. The complex was further characterized by Scanning Electron Microscopy (SEM) and Infrared Absorption Spectroscopy (FTIR). The membranes were manufactured at different pre-evaporation times and thicknesses and their morphology was analyzed by SEM and wettability through the contact angle. The results reveal the best cellulose acetate membrane produced in 30 sec and 1 mm thick, resulting in 55.58% ± 2.78 free fatty acids (FFA) in enzymatic hydrolysis in a membrane bioreactor and 100% rejection of oil in the membrane, using lipase in free form, in 4h. Finally, using the immobilized biocatalyst, the process with simultaneous separation resulted in 35.67% ± 1.02 of FFA and 0.82% ± 0.05 in the permeate, also with 100% oil rejection in the membrane. The products were evaluated by FTIR and triglycerides, diglycerides and monoglycerides were quantified using High Performance Liquid Chromatography (HPLC). After 5 cycles, the hydrolysis activity of the immobilized lipase remained at 75.62% ± 5.14. The study expands the understanding of enzyme immobilization, emphasizing the viability of graphene oxide as a support and the efficiency of simultaneous membrane separation of enzymatic hydrolysis, benefiting from efficiency and economy.