Produção, caracterização e imobilização de lipases e sua aplicação na síntese de ésteres alquílicos de ácidos graxos
Abstract
The use of industrial palm oil processing residues as a source of carbon and inducer for microbial lipase production can be an attractive way to add value to these residues and help reduce the costs of their production. In addition, the immobilization of these enzymes on solid supports is a way of improving their operational stability and facilitating their recovery and reuse in the process. The characteristics of the carrier and the immobilization technique employed can modulate the catalytic properties of lipases, in some cases altering their regio- and/or stereospecificities. In this context, this work had two main objectives: (i) to investigate the viability of the use of industrial palm oil residues as raw material for the production of lipase in different culture systems; (ii) to evaluate the effect of immobilization support and post-immobilization modification techniques on the activity, stability and specificity of immobilized lipase. Regarding lipase production, after the preliminary screening of 18 fungi from the Embrapa collection, the Aspergillus niger C strain was selected to evaluate lipase production by solid state (SSF) and submerged (SF) fermentation. The lipolytic activities achieved by SSF and SF of A. niger C were 15.41 and 10.46 IU/mL, respectively. The lipase produced by A. niger C by SSF showed maximum lipolytic activity at pH between 4.0 and 6.5 and at temperature between 37 and 55°C. This enzyme presented good stability at 60°C (half-life around 12 h) and greater specificity towards long carbon chain substrates, especially palm and sunflower oils. As for the immobilization step, the immobilization of a commercial lipase of Thermomyces lanuginosus (LTL) on different hydrophobic commercial supports and post-immobilization modification (glycosylation, hydroxylation and PEGylation) was first evaluated in order to verify the effect of the support and the post-immobilization modification technique on lipase activity, stability and specificity. Immobilization of LTL in Purolite C18 (support containing octadecyl groups) under different experimental conditions allowed the preparation of immobilized derivatives with different regiospecificities and reaction rates. Immobilization at pH 8.5 and 30°C in the presence of CTAB (cetyltrimethylammonium bromide) produced a derivative with perfect sn-1.3 regiospecificity. On the other hand, immobilization at pH 5.0 and 4°C in the absence of CTAB produced a nonspecific derivative which catalyzed the ethanolysis of acyl groups from the sunflower oil at sn-1 and sn-3 positions very quickly as well as at sn-2 position continuously. After the chemical modification of LTL adsorbed on Purolite C18 (Purolite-LTL) with PEG, the modified derivative showed high stability in an organic solvent free system (half-life of 6 days at 40ºC) and in hexane (100% activity after 20 days at 40°C). Subsequently, the immobilization of partially purified A. niger C lipase was evaluated on the supports Purolite C18 and octyl-silica (octyltriethoxysilane-modified silica). Immobilization yields were low (28-69%, in terms of activity), compared to those obtained with LTL immobilized on the same supports (approximately 100%). However, the derivative produced by the immobilization of A. niger C lipase on Purolite C18 (Purolite-LAN) showed similar performance to the Purolite-LTL derivative in the esterification of oleic acid with n-octanol, achieving oleic acid conversions of approximately 80% after 24 h of reaction. Therefore, this work stands out in an economic-environmental context, by the use of an agroindustrial residue of the processing of palm oil as a substrate for the production of an enzyme of great economic interest in different industrial sectors and by obtaining a competitive biocatalyst with a commercial lipase in important biotechnological applications.
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