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The enzyme β-galactosidase from Aspergillus oryzae has been immobilized in aminated (MANAE)-agarose beads via glutaraldehyde chemistry using different strategies. The immobilization on MANAE-supports was first assayed at different pH values (this gave different stabilities to the immobilized enzymes) and further modified with glutaraldehyde. Dramatic drops in activity were found, even using 0.1% ( v / v ) glutaraldehyde. The use of a support with lower activation permitted to get a final activity of 30%, but stability was almost identical to that of the just adsorbed enzyme. Next, the immobilization on pre-activated glutaraldehyde beads was assayed at pH 5, 7 and 9. At pH 7, full, rapid immobilization and a high expressed enzyme activity were accomplished. At pH 9, some decrease in enzyme activity was observed. Direct covalent immobilization of the enzyme was very slow; even reducing the volume of enzyme/support ratio, the yield was not complete after 24 h. The stability of the biocatalyst using pre-activated supports was about 4–6 folds more stable than that of the enzyme immobilized via ion exchange at pH 5, with small differences among them. Thus, the immobilization of the enzyme at pH 7 at low ionic strength on pre-activated glutaraldehyde supports seems to be the most adequate in terms of activity, stability and immobilization rate. ; We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). HadjerZaak thanks the Algerian Ministry of higher education and scientific research for the fellowships. The pre-doctoral fellowships for de Albuquerque (CNPq, Brazil) and Miss Peirce (Universita' degli Studi di Napoli Federico II) are also gratefully recognized. The help and suggestions of Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are gratefully recognized.

Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica , lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases. ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). The predoctoral fellowships for Rueda (Colciencias, Colombian Government and Becas Iberoamérica "Jóvenes Investigadores", Banco Santander) and dos Santos and Albuquerque (CNPq, Brazil) are also recognized. The authors wish to thank Ramiro Martínez (Novozymes, Spain) for kindly supplying some of the enzymes used in this research.