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

Ana Lúcia Ferrarezi, Daniele H. Pivetta, Gustavo Orlando Bonilla-Rodriguez, Roberto da Silva, José Manuel Guisan, Eleni Gomes, Benevides Costa Pessela

Department of Biology, Laboratory of Applied Biochemistry and Microbiology, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil.
Department of Biotechnology and Food Microbiology, Research Institute for Food Science, CIAL-CSIC, CalleNicolás Cabrera 9, Campus UAM, Madrid, Spain.
Department of Chemistry and Environmental Sciences, IBILCE-UNESP, S?o José do Rio Preto SP, Brazil.
Department of Enzyme Biocatalysis, Institute of Catalysis and Petrochemistry, CSIC, Campus UAM, Cantoblanco, Madrid, Spain.

DOI: 10.4236/aer.2013.14009                

Abstract

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.

Keywords

Enzyme Immobilization; Solid State Fermentation; Purification; n-3 Polyunsatured Fatty Acids; Rhizomucor pusillus

Conflicts of Interest

The authors declare no conflicts of interest.

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