doi:10.1021/bm500333vengVelasco-Lozano, SusanaLópez-Gallego, FernandoVázquez-Duhalt, RafaelMateos-Díaz, Juan C.Guisán, José M.Favela-Torres, ErnestoCarrier-Free Immobilization of Lipase from <i>Candida rugosa</i> with Polyethyleneimines by Carboxyl-Activated Cross-LinkingART-2014-147686Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethyleneimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs’ size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50°C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.2014http://zaguan.unizar.es/record/16815810.1021/bm500333vhttp://zaguan.unizar.es/record/168158oai:zaguan.unizar.es:168158BIOMACROMOLECULES 15, 5 (2014), 1896-1903All rights reservedhttp://www.europeana.eu/rights/rr-f/info:eu-repo/semantics/openAccess