PARAMETERS FOR THE IMMOBILIZATION OF A LIPASE PRODUCED BY AN AMAZONIAN FILAMENTOUS FUNGUS ON A HYDROPHOBIC SUPPORT ACTIVATED WITH GLUTARALDEHYDE

Immobilization positively affects enzyme stability, since the immobilized lipase exhibits higher activity compared to the soluble lipase. This work aimed to immobilize a lipase from an Amazonian filamentous fungus isolated from bacaba (Oenocarpus bacaba), a typical Amazonian fruit, on glutaraldehyde-activated polyhydroxybutyrate (GLU-PHB) for further essential fatty acids production. The soluble enzyme was produced by submerged cell cultivation in an orbital shaker at 30 °C and 200 rpm for 168 h, inoculating 1x10^7 spores/mL in a synthetic culture medium (pH 5.5) formulated with 5.5 g/L of K2HPO4, 15 g/L of KHPO4, 0.5 g/L of MgSO4.7H2O, 1.0% (m/v) of olive oil and 0.2% of yeast extract (m/v). The crude extract was used for the immobilization assays. PHB particles of 1.2 mm were washed with 95% (v/v) ethanol p.a at the proportion of 1:10 (w/v) at 120 rpm and 30 ºC for 6 h. Subsequently, the biopolymer was activated in the glutaraldehyde solution (25% in water), sodium phosphate buffer at 5 mmol/L, pH 7.0, at 120 rpm and 30 ºC for 18 h. GLU-PHB was vacuum-filtered and dried in a desiccator for 48 h. The immobilization assays were performed with 1 g of GLU-PHB in 10 mL of filtered crude extract at 35 °C, 175 rpm and pH 5.5 for 8 h. The enzymatic reactions were conducted at 175 rpm and 37 ºC for 5 minutes, using olive oil as substrate. The hydrolytic activity of the soluble and immobilized enzyme was quantified by titration with NaOH at 0.05 mol/L. The immobilization yield of the lipase on GLU-PHB was 21.4%, while its recovery activity was about 94.34%. These results suggest the lipase was satisfactorily immobilized on GLU-PHB due to the hydrophobic nature of this organic support and, therefore, the enzymatic derivates obtained are promising biocatalysts for essential fatty acids production in heterogeneous reaction systems.