Free and Substrate-Immobilised Lipases from Fusarium verticillioides P24 as a Biocatalyst for Hydrolysis and Transesterification Reactions.

Fungal enzymes are widely used in technological processes and have some interesting features to be applied in a variety of biosynthetic courses. Here, free and substrate-immobilised lipases from Fusarium verticillioides P24 were obtained by solid-state fermentation using wheat bran as substrate and fungal carrier. Based on their hydrolytic and transesterification activities, the lipases were characterised as pH-dependent in both reactions, with higher substrate conversion in an alkaline environment. Thermally, the lipases performed well from 30 to 45 °C, being more stable in mild conditions. Organic solvents significantly influenced the lipase selectivity using different vegetable oils as fatty acid source. Omega(ω)-3 production in n-hexane achieved 45% using canola oil, against ≈ 18% in cyclohexane. However, ω-6 production was preferably produced for both solvents using linseed oil with significant alterations in the yield (≈ 79% and 49% for n-hexane and cyclohexane, respectively). Moreover, the greatest enzyme selectivity for ω-6 led us to suppose a lipase preference for the Sn1 position of the triacylglycerol. Lastly, a transesterification reaction was performed, achieving 90% of ester conversion in 72 h. This study reports the characterisation and use of free and substrate-immobilised lipases from Fusarium verticillioides P24 as an economic and efficient method for the first time.

Ethyl esters production catalyzed by immobilized lipases is influenced by n-hexane and ter-amyl alcohol as organic solvents.

Lipase stability in organic solvent is crucial for its application in many biotechnological processes as biocatalyst. One way to improve lipase's activity and stability in unusual reaction medium is its immobilization on inert supports. Here, lipases from different sources and immobilized through weak chemical interactions on hydrophobic and ionic supports had their transesterification ability dramatically dependent on the support and also on the solvent that had been used. The ethanolysis of sardine oil was carried out at the presence of cyclohexane and tert-amyl alcohol, in which Duolite A568-Thermomyces lanuginosa lipase derivative achieved 49% of ethyl esters production after 24 h in cyclohexane. The selectivity of immobilized lipases was also studied and, after 3 h of synthesis, the reaction with Duolite A568-Thermomyces lanuginosa derivative in cyclohexane produced 24% ethyl ester of eicosapentaenoic acid and 1.2% ethyl ester of docosahexaenoic acid, displaying a selectivity index of 20 times the ethyl ester of eicosapentaenoic acid. Different derivatives of Candida antarctica lipases fraction B (CALB) and phospholipase Lecitase® Ultra (Lecitase) were also investigated. Along these lines, a combination between these factors may be applied to improve the activity and selectivity of immobilized lipases, decreasing the total cost of the process.

Critical Role of Different Immobilized Biocatalysts of a Given Lipase in the Selective Ethanolysis of Sardine Oil.

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLL-Sepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLL-Duolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA.

Hydrophobic adsorption in ionic medium improves the catalytic properties of lipases applied in the triacylglycerol hydrolysis by synergism.

It is known that lipases may have their catalytic properties improved by the action of some salts or by the adsorption on hydrophobic supports. However, what we present in this work is more than that: we evaluate the combination of these two factors of hyperactivation of lipases from Acremonium-like ROG 2.1.9, a study that has not been done so far. This work proves that a synergistic effect occurs when the lipases are immobilized on hydrophobic supports at the presence of sodium chloride and are applied in triacylglycerol hydrolysis. This assay made it possible to achieve the highest hyperactivation of 500 % with the lipases immobilized on Phenyl-Sepharose and applied with 0.1 M of sodium chloride. Besides this positive effect on enzyme activity, the use of these two factors led to the thermal stability increasing of the immobilized lipases. For this derivative, the recovered activity was approximately 85 % after 6 h incubated at 55 °C and 1.0 M of the sodium chloride against 50 % of the same derivative without this salt. Furthermore, others assays were performed to prove the evidences about the synergistic effect, showing a promising method to improve the catalytic properties of the lipases from Acremonium-like ROG 2.1.9.