Microalgal biomass is a sustainable source of bioactive lipids with omega-3 fatty acids. The efficient extraction of neutral and polar lipids from microalgae requires alternative extraction methods, frequently combined with biomass pretreatment. In this work, a combined ultrasound and enzymatic process using commercial enzymes Viscozyme, Celluclast, and Alcalase was optimized as a pretreatment method for Nannochloropsis gaditana, where the Folch method was used for lipid extraction. Significant differences were observed among the used enzymatic pretreatments, combined with ultrasound bath or probe-type sonication. To further optimize this method, ranges of temperatures (35, 45, and 55 °C) and pH (4, 5, and 8) were tested, and enzymes were combined at the best conditions. Subsequently, simultaneous use of three hydrolytic enzymes rendered oil yields of nearly 29%, showing a synergic effect. To compare enzymatic pretreatments, neutral and polar lipids distribution of Nannochloropsis was determined by HPLC–ELSD. The highest polar lipids content was achieved employing ultrasound-assisted enzymatic pretreatment (55 °C and 6 h), whereas the highest glycolipid (44.54%) and PE (2.91%) contents were achieved using Viscozyme versus other enzymes. The method was applied to other microalgae showing the potential of the optimized process as a practical alternative to produce valuable lipids for nutraceutical applications.

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Oxidases catalyze selective oxidations by using molecular oxygen as an oxidizing agent. This process promotes the release of hydrogen peroxide, an undesirable byproduct. The instantaneous elimination of hydrogen peroxide can be achieved by co-immobilization and co-localization of the oxidase and an auxiliary catalase inside the porous structure of solid support. In this paper, we proposed that catalase from Bordetella pertussis fused with a small domain (Zbasic) as an excellent auxiliary enzyme. The enzyme had a specific activity of 23 U/mg, and this was almost six-fold higher than the one of the commercially available catalases from bovine liver. The Zbasic domain was fused to the four amino termini of this tetrameric enzyme. Two domains were close in one hemisphere of the enzyme molecule, and the other two were close in the opposite hemisphere. In this way, each hemisphere contained 24 residues with a positive charge that was very useful for the purification of the enzyme via cationic exchange chromatography. In addition to this, each hemisphere contained 10 Lys residues that were very useful for a rapid and intense multipoint covalent attachment on highly activated glyoxyl supports. In fact, 190 mg of the enzyme was immobilized on one gram of glyoxyl-10% agarose gel. The ratio catalase/oxidase able to instantaneously remove more than 93% of the released hydrogen peroxide was around 5–6 mg of catalase per mg of oxidase. Thirty milligrams of amine oxidase and 160 mg of catalase were co-immobilized and co-localized per gram of glyoxyl-agarose 10BCL (10% beads cross-linked) support. This biocatalyst eliminated biogenic amines (putrescine) 80-fold faster than a biocatalyst of the same oxidase co-localized with the commercial catalase from bovine liver.