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Partial purification,immobilization and preliminary biochemical characterization of lipases from Rhizomucor pusillus

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Advances in Enzyme Research 2013年第4期1卷 79-90页

作者： Ana Lucia Ferrarezi Daniele H.Pivetta Gustavo Orlando Bonilla-Rodriguez Roberto da Silva Jose Manuel Guisan Eleni Gomes Benevides Costa Pessela Department of Biology Laboratory of Applied Biochemistry and MicrobiologyIBILCE-UNESPSao Jose do Rio Preto SPBrazil Department of Chemistry and Environmental Sciences IBILCE-UNESPSao Jose do Rio Preto SPBrazil Department of Enzyme Biocatalysis Institute of Catalysis and PetrochemistryCSICCampus UAMCantoblancoMadridSpain Department of Biotechnology and Food Microbiology Research Institute for Food ScienceCIAL-CSICCalleNicolás Cabrera 9Campus UAMMadridSpain

Lipases have important applications in biotechnological processes, motivating us to produce, purify, immobilize and perform a biochemical characterization of the lipase from Rhizomucor pusillus. The fungus was cultivated by solid state fermentation producing lipolytic activity of about 0.5 U/mL(4U/g). A partial purification by gel filtration chromatography in Se-phacryl S-100 allowed obtaining a yield of about 85% and a purification factor of 5.7. Our results revealed that the purified enzyme is very stable with some significant differences in its properties when compared to crude extract. The crude enzyme extract has an optimum pH and temperature of 7.5 ° C and 40 ° C, respectively. After purification, a shift of the optimum pH from 7 to 8 was observed, as well as a rise in optimumtemperature to 60 ° C and an increase in stability. The enzyme was immobilized on CnBr-Agarose and Octyl-Agarose supports, having the highest immobilization yield of 94% in the second resin. The major advantage of immobilization in hydrophobic media such as Octyl is in its hyper activation, which in this case was over 200%, a very interesting finding. Another advantage of this type of immobilization is the possibility of using the derivatives in biotechnological applications, such as in oil enriched with omega-3 as the results obtained in this study display the hydrolysis of 40% EPA and 7% DHA from sardine oil, promising results compared to the literature.

关键词： Enzyme Immobilization Solid State Fermentation Purification n-3 polyunsatured fatty acids Rhizomucor pusillus

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