This paper shows the coimmobilization of β-galactosidase from Aspergillus oryzae (β-gal) and lipase B from Candida antarctica (CALB). The combi-biocatalyst was designed in a way that permits an optimal immobilization of CALB on octyl-agarose (OC) and the reuse of this enzyme after β-gal (an enzyme with lower stability and altogether not very stabilized by multipoint covalent attachment) inactivation, both of them serious problems in enzyme co-immobilization. With this aim, OC-CALB was coated with polyethylenimine (PEI) (this treatment did not affect the enzyme activity and even improved enzyme stability, mainly in organic medium). Then, β-gal was immobilized by ion exchange on the PEI coated support. We found that PEI can become weakly adsorbed on an OC support, but the adsorption of PEI to CALB was quite strong. The immobilized β-gal can be desorbed by incubation in 300 mM NaCl. Fresh β-gal could be adsorbed afterwards, and this could be repeated for several cycles, but the amount of PEI showed a small decrease that made reincubation of the OC-CALB-PEI composite in PEI preferable in order to retain the amount of polymer. CALB activity remained unaltered under all these treatments. The combi-catalyst was submitted to inactivation at 60 °C and pH 7, conditions where β-gal was rapidly inactivated while CALB maintained its activity unaltered. All β-gal activity could be removed by incubation in 300 mM NaCl, however, SDS analysis showed that part of the enzyme β-gal molecules remained immobilized on the OC-CALC-PEI composite, as the inactivated enzyme may become more strongly adsorbed on the ion exchanger. Full release of the β-gal after inactivation was achieved using 1 M NaCl and 40 °C, conditions where CALB remained fully stable. This way, the proposed protocol permitted the reuse of the most stable enzyme after inactivation of the least stable one. It is compatible with any immobilization protocol of the first enzyme that does not involve ion exchange as only reason for enzyme immobilization. © 2016 The Royal Society of Chemistry

27The effect of an additive on enzyme stability use to be considered an intrinsic feature of 28 the lipase. However, in this paper we have found that the effect of additive on enzyme stability 29 is depended on the immobilization protocol. After assaying the effects of diverse chloride salts 30 with different cations on different lipases activity, no relevant effect was detected. Free enzymes 31 or the covalently immobilized enzymes are not stabilized by these cations for any of the studied 32 lipases. However, Mn 2+ and Ca 2+ (at a concentration of 5 mM) are able to greatly stabilize the 33 lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present 34 during the inactivation, but only if the enzymes are immobilized on octyl-agarose (stabilization 35 factor ranging from 20 to 50). The effect was only detected using more than 2.5 mM of the 36 cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action. 37The stabilization seemed to be based on a specific mechanism, and required to have the 38 recognition sites saturated by the cations. Mg 2+ has no effect on enzyme stability for both 39 enzymes, but it is able to suppress the stabilization promoted by the other two cations using 40 CRL; while it has no effect on the cation stabilization when using RML. 41 This is the first report of a cation induced enzyme stabilization effect that depends on the 42 lipase immobilization protocol. 43 44

Abstract: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.

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