IMMOBILIZATION OF CANDIDA VISWANATHII LIPASE ON MODIFIED HYDROPHOBIC RESIN

Lipases belong to the hydrolase family and are widely studied in biotechnology due to their potential in various industrial sectors, particularly in the food industry, acting in the hydrolysis and synthesis of lipids, enabling the modification of oils and fats of nutritional and technological interest. The use of soluble enzymes presents limitations related to stability and reuse, motivating immobilization strategies on supports. The objective of this work was to immobilize Candida viswanathii lipase on Immobead 150P resin through an experimental design, varying temperature (10 °C; 17.5 °C and 25 °C) and pH (4.0; 5.5 and 7.0) to define the process conditions and analyze the effects on immobilization yield and recovered activity. The enzyme was obtained by submerged fermentation and immobilization was performed on Immobead treated with epichlorohydrin or with ethylenediamine and epichlorohydrin. During the assays, supernatant aliquots were collected from 0 to 11 h and 24 h, and total protein was determined by the Lowry method. Enzymatic activity was evaluated with p-nitrophenyl palmitate and immobilization yield and recovered activity were calculated. The support treated only with epichlorohydrin showed equilibrium times between 10 and 24 h and the highest immobilization yield of 83% (pH 4.0 and 10 °C), but with low recovered activity, reaching at most 10%. The formation of a dense layer of epoxy groups on the support surface may restrict the mobility and functionality of the enzyme. Immobilization resulted in a specific activity of 12.1 U·mg⁻¹, compared to 11.3 U·mg⁻¹ of the crude extract, indicating selective binding of lipase molecules, while contaminants reacted in smaller amounts. On the support modified with EDA and epichlorohydrin, equilibrium times were shorter (4 to 6 h), yield ranged from 40% to 69%, and recovered activity was higher, reaching 16%, with a specific activity of 10.4 U·mg⁻¹. Epoxy supports tend to show low recoveries with highly glycosylated enzymes, due to the protection of active groups by the polysaccharide fraction, as observed for C. viswanathii lipase. Diffusion limitations, the presence of contaminants from the crude extract and the formation of dimeric aggregates may reduce the interaction of the enzyme with the substrate, explaining the low recovery observed. The immobilization of C. viswanathii lipase on modified Immobead 150P showed immobilization yield and recovered activity dependent on pH, temperature and support treatment, highlighting the need for optimization and future characterizations.