Monday, May 5, 2008
12-17

Purification and immobilization of lipase from Penicillium simplicissimum by selective adsorption on hydrophobic supports

Aline G. Cunha1, Gloria Fernández-Lorente2, Melissa L. E. Gutarra1, Juliana Vaz Bevilaqua3, Lucia M. C. Paiva1, Roberto Fernández-Lafuente2, Jose M. Guisán2, and Denise M.G. Freire1. (1) Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, (2) Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain, (3) Cenpes, Petrobras, Rio de Janeiro, Brazil

Lipases (glycerol ester hydrolases E.C. 3.1.1.3) consist of a class of enzyme that has great importance as biocatalysts applied to traditional organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures and organic solvents and high stereoselectivity. The aim of the present work was to purify and simultaneously immobilize the pool of lipases produced by P. simplicissimum (PSL), a filamentous fungi isolated from Pantanal soil in Brazil. PSL was separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, octyl-agarose) at low ionic strength. After immobilization, a hyperactivation of these fractions was observed in the range of 131% to 1133%. This phenomenon probably occurs because the immobilization of the lipase is in active open form onto hydrophobic supports. Those fractions, showed different thermal stability, different specificity and enantioselectivity upon some substrates, with enantioselectivity from 1 to 7.9 for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid. Prochiral diethyl phenylmalonate were partially hydrolyzed to the corresponding chiral monoesters by the different immobilized PSL fractions. Those fractions neither catalyzed the hydrolysis of the monoesters nor produced the final achiral di-carboxylic acid yielding chiral monoesters, with asymmetry factors from 11.8 to 16.4 depending on the immobilized PSL fractions used. Partial purification of a microbial crude extract through sequential adsorption methodology demonstrated to be an efficient strategy to obtain new biocatalysts with different degrees of enantioselectivity.

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