Immobilization and Purification of Heavy-Metal Resistant Lipases of Hypocrea pseudokoningii Produced in Solid-State Fermentation

Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacylglycerols into mono- and diacylglycerols and free fatty acids. This study investigated the production of lipase by Hypocrea pseudokoningii under solid-state fermentation (SSF), followed by its immobilization, purification, and biochemical characterization. Maximum activity was achieved using wheat fiber after 168 h of cultivation. Supplementation with oils enhanced production, particularly palm oil (315U; 1.58-fold increase) and soybean oil (Glycine max) (298U; 1.49-fold increase). The addition of micronutrients further improved yields, with Khanna (445U) and Vogel (400U) salts promoting more than a two-fold increase in activity. Immobilization on Octyl-Sepharose significantly altered the enzyme’s properties. The free lipase exhibited optimal activity at 45 °C and pH 4.5–5.5, while the immobilized enzyme showed maximum activity at 35–40 °C and pH 5.5. Thermal stability was notable enhanced: the free lipase had a half-life of 10 min at 50 °C, whereas the immobilized enzyme remained stable for 60 min and retained over 30% activity at 70 °C. Both the free and immobilized forms were stable across a broad pH range (4.0–10.0), maintaining more than 70% residual activity. The enzyme was stabilized by Tween 80 but inhibited by SDS. It was activated by Ca2+ and showed resistance to Pb2+, Zn2+, and Cu2+. Hydrolytic assays revealed murumuru (Astrocaryum murumuru), cupuaçu (Theobroma grandiflorum), and soybean oils as preferred substrates. TLC confirmed the formation of mono- and diglycerides, as well as the presence of fatty acids.