RESUMEN
The lipase from Pseudomonas fluorescens (PFL) has been immobilized on glyoxyl-octyl agarose and compared to the enzyme immobilized on octyl-agarose. Thus, PFL was immobilized at pHâ¯7 on glyoxyl-octyl support via lipase interfacial activation and later incubated at pHâ¯10.5 for 20â¯h before reduction to get some enzyme-support covalent bonds. This permitted for 70% of the enzyme molecules to become covalently attached to the support. This biocatalyst was slightly more stable than the octyl-PFL at pHâ¯5, 7 and 9, or in the presence of some organic solvents (stabilization factor no higher than 2). The presence of phosphate anions produced enzyme destabilization, partially prevented by the immobilization on glyoxyl-octyl (stabilization factor became 4). In contrast, the presence of calcium cations promoted a great PFLstabilization, higher in the case of the glyoxyl-octyl preparation (that remained 100% active when the octyl-PFL preparations had lost 20% of the activity). However, it is in the operational stability where the new biocatalyst showed the advantages: in the hydrolysis of 1â¯M triacetin in 60% 1.4 dioxane, the octyl biocatalyst released >60% of the enzyme in the first cycle, while the covalently attached enzyme retained its full activity after 5 reaction cycles.
Asunto(s)
Proteínas Bacterianas/química , Enzimas Inmovilizadas/química , Glioxilatos/química , Lipasa/química , Pseudomonas fluorescens/enzimología , Sefarosa/química , Estabilidad de Enzimas , Concentración de Iones de HidrógenoRESUMEN
A novel heterofunctional support for enzyme immobilization, chitosan-divinyl sulfone, was assessed in this study. The activation of chitosan with DVS was carried out at three different pHs (10.0, 12.5 and 14.0) and a Candida antarctica Lipase B (CALB) was selected as the model enzyme. After immobilization, the biocatalysts were incubated under alkaline conditions in a buffer to facilitate the multipoint covalent attachment, followed by incubation in ethylenediamine (EDA) aiming at blocking the remaining reactive groups. The highest thermal stability was obtained when pHâ¯10.0 was used during support activation. These results were shown to be better than those obtained when using glutaraldehyde as the support-activating reagent. Subsequently, the immobilization pH was investigated (5.0, 7.0 and 10.0) prior to alkaline incubation, with the highest enzyme stability levels found at pHâ¯10.0. Finally, the selected biocatalyst was used in the hydrolysis of ethyl hexanoate and presented an activity of 14,520.37â¯U/g of immobilized lipase at pHâ¯5.0. These results show that chitosan activated with divinyl sulfone is a very promising support for enzyme immobilization and the proposed protocol is able to successfully improve enzyme stability.