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In this study, Penicillium sp. section Gracilenta CBMAI 1583 was used to produce lipase under submerged conditions. The enzyme was purified and the biochemical properties of both the crude and purified enzymes were evaluated. Maximum lipase production (1.62 U mL−1) was obtained using olive oil 0.5% (w v−1) after 72 h of cultivation, representing a 90% increase in the lipase initially produced. The enzyme was purified using hydrophobic interaction chromatography (phenyl Sepharose) under conditions which allowed its interfacial activation. The partially purified sample showed an enzyme with esterase activity (65.4 kDa) on α- and β-naphthyl acetate and other with lipase activity (52.9 kDa) on octyl oleate. Optimum activity of crude and purified lipase was observed at pH 4.0 and 70 °C. The purified lipase was activated by NaCl, BaCl2, NH4Cl, MnSO4 and MgSO4; it also presented high stability in organic solvents such as hexane, 2.2.4-trimethylpentane, acetone, DMSO and toluene. Maximum enzyme activity was observed with p-nitrophenyl decanoate as substrate; and the enzyme kinetics showed to be directly affected by Triton X-100. The enzyme shows potential application in processes that operate in acid pH, such as treatment of dairy and industry effluents, resolution of esters in the pharmaceutical industry or in the food industry, as well in synthesis reaction under non-aqueous conditions., The authors gratefully acknowledge to São Paulo Research Foundation - FAPESP, Brazil, for the scholarship granted to the first author and Spanish Ministry of Science and Innovation - MICINN, Spain (Project BIO-2012–36861).

Different immobilized derivatives of two lipases were tested as catalysts of the synthesis of ethyl esters of omega-3 fatty acids during the ethanolysis of sardine oil in solvent-free systems at 25 °C. Lipases from Thermomyces lanuginosus (TLL) and Lecitase Ultra (a phospholipase with lipolytic activity) were studied. Lipases were adsorbed on hydrophobic Sepabeads C18 through the open active center and on an anion-exchanger Duolite with the active center exposed to the reaction medium. TLLSepabeads derivatives exhibit a high activity of 9 UI/mg of immobilized enzyme, and they are 20-fold more active than TLLDuolite derivatives and almost 1000-fold more active than Lipozyme TL IM (the commercial derivative from Novozymes). Lecitase-Sepabeads exhibit a high selectivity for the synthesis of the ethyl ester of EPA that is 43-fold faster than the synthesis of the ethyl ester of DHA., This work was sponsored by the Spanish Ministry of Science and Innovation (Projects AGL-2009-07526 and BIO2012-36861). We gratefully recognize the Spanish Ministry of Science and Innovation for the “Ramon y Cajal> contract for G.F.-L. and for the FPI contract to S.M.-P. We also thank Fundaca̧o de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) for granting the 284 scholarship to D.F.M.T.i (BEPE Process 2014/04925-1).

Made available in DSpace on 2018-12-11T17:11:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties. Department of Biochemistry and Microbiology Biosciences Institute Universidade Estadual Paulista (UNESP) Instituto de Investigación en Ciencias de la Alimentación (CIAL) CSIC-UAM Instituto de Catálisis y Petroleoquímica (ICP) CSIC-UAM Pharmacy and Biotechnology Department School of Biomedical Sciences Universidad Europea Department of Biochemistry and Microbiology Biosciences Institute Universidade Estadual Paulista (UNESP)