Integrated Process for Schizochytrium Oil Extraction, Enzymatic Modification of Lipids and Concentration of DHA Fatty Acid Esters Using Alternative Methodologies.

Marine microalgae Schizochytrium sp. have a high content of docosahexaenoic acid (DHA), an omega-3 fatty acid that is attracting interest since it prevents certain neurodegenerative diseases. The obtention of a bioactive and purified DHA fatty acid ester using a whole-integrated process in which renewable sources and alternative methodologies are employed is the aim of this study. For this reason, lyophilized Schizochytrium biomass was used as an alternative to fish oil, and advanced extraction techniques as well as enzymatic modification were studied. Microalgal oil extraction was optimized via a surface-response method using pressurized liquid extraction (PLE) obtaining high oil yields (29.06 ± 0.12%) with a high concentration of DHA (51.15 ± 0.72%). Then, the enzymatic modification of Schizochytrium oil was developed by ethanolysis using immobilized Candida antarctica B lipase (Novozym® 435) at two reaction temperatures and different enzymatic loads. The best condition (40 °C and 200 mg of lipase) produced the highest yield of fatty acid ethyl ester (FAEE) (100%) after 8 h of a reaction attaining a cost-effective and alternative process. Finally, an enriched and purified fraction containing DHA-FAEE was obtained using open-column chromatography with a remarkably high concentration of 93.2 ± 1.3% DHA. The purified and bioactive molecules obtained in this study can be used as nutraceutical and active pharmaceutical intermediates of marine origin.

Enzymatic synthesis of mono- and disubstituted phospholipids by direct condensation of oleic acid and glycerophosphocholine with immobilized lipases and phospholipase.

Lysophospholipids which contain polyunsaturated fatty acids play a key role in food and cosmetic industries because of their bioactivity. Therefore, the formation of mono- and disubstituted phospholipids is quite interesting as they could be used for the formation of different natural liposomes. Using immobilized derivatives of lipases and phospholipases, the esterification of oleic acid with glycerophosphocholine (GPC) has been studied. Thus, derivatives were quite active in completely anhydrous media and in solvent-free reaction systems where the reaction takes place. CALB biocatalyst was able to successfully form oleoyl-LPC at 60 °C in the presence of 30 % butanone, where the synthesis rate was 100 times higher than in the absence of solvents at 40 °C. On the other hand, the best synthesis rate for dioleoyl-PC was achieved with immobilized Lecitase in a solvent-free process at 60 °C, an 83 % synthesis yield was achieved with an initial synthesis rate of 4.32 mg/mL × h × g.

Cross-Linked Enzyme Aggregates and Their Application in Enzymatic Pretreatment of Microalgae: Comparison Between CLEAs and Combi-CLEAs.

Carrier-free immobilization is a key process to develop efficient biocatalysts able to catalyze the cell wall degradation in microalgae where the traditional solid supports cannot penetrate. Thus, the insolubilization of commercial Celluclast®, Alcalase®, and Viscozyme® enzymes by carrier-free immobilization and their application in microalgae pretreatment was investigated. In this study, different precipitants at different ratios (ethanol, acetone, and polyethylene glycol 4000) were tested in the first part of the method, to establish the precipitation conditions. The screening of the best precipitant is needed as it depends on the nature of the enzyme. The best results were studied in terms of immobilization yield, thermal stability, and residual activity and were analyzed using scanning electron microscopy. Moreover, a novel strategy was intended including the three enzymes (combi-CLEAs) to catalyze the enzymatic degradation of Nannochloropsis gaditana microalgal cell wall in one pot. The carrier-free immobilized derivatives were 10 times more stable compared to soluble enzymes under the same. At the best conditions showed its usefulness in the pretreatment of microalgae combined with ultrasounds, facilitating the cell disruption and lipid recovery. The results obtained suggested the powerful application of these robust biocatalysts with great catalytic properties on novel and sustainable biomass such as microalgae to achieve cost-effective and green process to extract valuable bioactive compounds.

Combination of Synergic Enzymes and Ultrasounds as an Effective Pretreatment Process to Break Microalgal Cell Wall and Enhance Algal Oil Extraction.

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.

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation.

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the "capture" and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is "fixed" on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.

A mild intensity of the enzyme-support multi-point attachment promotes the optimal stabilization of mesophilic multimeric enzymes: Amine oxidase from Pisum sativum.

Stabilization of dimeric enzymes requires the stabilization of the quaternary structure as well as the 3D one. Both subunits may be easily immobilized on a highly activated support. Additional stabilization of the 3D structure may be achieved via multipoint covalent attachment (MCA) on highly activated supports. In the case of monomeric enzymes or thermophilic dimeric ones, the optimal stabilization is obtained via the most intense MCA and it is associated to a small loss of catalytic activity. However, in the case of mesophilic enzymes, a very intense MCA of both subunits may promote negative effects, e.g., associated to distortions of the assembly between subunits and a subsequent very important loss of catalytic activity. A dimeric mesophilic amine oxidase from P.sativum was stabilized by MCA on glyoxyl-agarose. Both subunits were covalently immobilized on the support through the region with the highest density in Lys residues. In addition to that, an interesting activity/stabilization binomial was obtained after only 3 h of enzyme-support multiinteraction (50 % of activity/350 fold stabilization). However, after 24 h of enzyme-support multi-interaction this binomial activity-stabilization decreased down to 30/150. A moderate multiinteraction seems to be the optimal strategy for immobilization-stabilization of mesophilic dimeric enzymes and it promotes moderate losses of activity and interesting stabilizations against the combined effect of heat, acid pH and ethanol. The control of the intensity of enzyme-support multi-interactions becomes now strictly necessary.

Co-localization of oxidase and catalase inside a porous support to improve the elimination of hydrogen peroxide: Oxidation of biogenic amines by amino oxidase from Pisum sativum.

Diamine oxidase (DAO) from Pisum sativum is an enzyme that catalyzes the degradation of biogenic amines (BA) present in wine, producing harmless aldehydes and hydrogen peroxide (H2O2). H2O2 promotes a rapid inactivation of the immobilized enzyme. At first glance, co-immobilization of DAO and catalase (CAT) could improve the elimination of the released hydrogen peroxide. Two different co-immobilized derivatives were prepared: (a) both enzymes co-localized and homogeneously distributed across the whole structure of a porous support, and (b) both enzymes we de-localized inside the porous support: DAO immobilized on the outer part of the porous support and catalase immobilized in the inner part. Co-localized derivatives were seven-fold more effective than de-localized ones for the elimination of hydrogen peroxide inside the porous support. In addition to that, the degradation of putrescine by DAO was three-fold more rapid when using both co-localized enzymes. The optimal co-localized derivative (containing 1.25 mg of DAO plus 25 mg of CAT per g of support) promoted the instantaneous elimination of 91% H2O2 released inside the porous support during putrescine oxidation. This optimal derivative preserves 92% of activity after three reaction cycles and DAO immobilized without catalase only preserves 41% of activity. Co-localization seems to be the key strategy to immobilize two sequential enzymes. When enzymes are immobilized in close proximity to each other in a co-localized pattern, the generation of byproducts as H2O2 is strongly reduced.