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Different strategies to immobilize lipase from Geotrichum candidum: kinetic and thermodynamic studies

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Matheus M. Ferreiraa, Fernanda L. B. Santiagob, Nádia A. G. da Silvab, Jaine H. H. Luizb, Roberto Fernandéz-Lafuentec, Adriano A. Mendesb, Daniela B. Hirataa,b*
aPostgraduate Program in Chemical Engineering, Federal University of Alfenas, 37715- 400 Poços de Caldas, MG, Brazil.
bInstitute of Chemistry, Federal University of Alfenas, 37130-000 Alfenas, MG, Brazil.
cDepartamento de Biocatálisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC,
Cantoblanco, 28049 Madrid, Spain.
The use of different strategies to immobilize lipase from Geotrichum candidum (GCLI) produced through submerged fermentation was evaluated in order to obtain a biocatalyst with a high stability and activity. Crude and purified GCLI were immobilized by different protocols such as interfacial activation, ionic adsorption and multipoint  covalent attachment. The biocatalyst prepared using purified GCLI immobilized on MANAE-agarose exhibited the highest hydrolytic activity (45.40 ± 0.19 IU/g of support) among all the supports used, therefore it was selected for kinetic and thermodynamic studies and the influence of the pH and the protein loading was investigated. The maximum adsorption capacity of 4.51 ± 0.21 mg/g of support was observed at pH 7.0 and 25 °C with a hydrolytic activity of 81.10 ± 2.11 IU/g. GCLI immobilization on MANAE-agarose also reduced the apparent activation energy by 50% compared to the soluble enzyme and increased the amount of product formed in hydrolysis reactions, which was verified by calculating the apparent kinetic parameters Vmax (46.30 IU/mL) and Km (261.77 mmol/L). All thermodynamic apparent parameters determined from thermal stability assays showed that immobilized GCLI allowed a great increase in thermostability. Also, the prepared biocatalyst was able to hydrolyze  methyl phenylacetate better than methyl mandelate
Keywords:immobilization; lipases, Geotrichum, thermodynamic parameters
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