Jose J. Virgen-Ortíz

Effect of protein load on stability of immobilized enzymes

Different lipases have been immobilized on octyl agarose beads at 1mg/g and at maximum loading, via physical interfacial activation versus the octyl layer on the support. The stability of the preparations was analyzed. Most biocatalysts had the expected result: the apparent stability increased using the highly loaded preparations, due to the diffusional limitations that reduced the initial observed activity. However, lipase B from Candida antarctica (CALB) was significantly more stable using the lowly loaded preparation than the maximum loaded one. This negative effect of the enzyme crowding on enzyme stability was found in inactivations at pH 5, 7 or 9, but not in inactivations in the presence of organic solvents. The immobilization using ethanol to reduce the immobilization rate had no effect on the stability of the lowly loaded preparation, while the highly loaded enzyme biocatalysts increased their stabilities, becoming very similar to that of the lowly loaded preparation. Results suggested that CALB molecules immobilized on octyl agarose may be closely packed together due to the high immobilization rate and this produced some negative interactions between immobilized enzyme molecules during enzyme thermal inactivation. Slowing-down the immobilization rate may be a solution for this unexpected problem.

Development of simple protocols to solve the problems of enzyme coimmobilization. Application to coimmobilize a lipase and a β-galactosidase

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.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports.

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst.

Evaluation of different commercial hydrophobic supports for the immobilization of lipases: tuning their stability, activity and specificity

Five different commercial supports (Lifetech™ ECR1061M (styrene/methacrylic polymer), Lifetech™ ECR8804M (octadecyl methacrylate), Lifetech™ ECR8806M (octadecyl methacylate), Lifetech™ ECR1090M (styrene) and Lifetech™ ECR1030M (DVB/methacrylic polymer)) were compared to octyl agarose in their performance in the immobilization of four different lipases (from Rhizomucor miehie (RML), from Thermomyces lanuginosus (TLL) and the forms A and B from Candida antarctica, (CALA and CALB)) and of the phospholipase Lecitase Ultra™ (LU). The new enzymatic derivatives were evaluated and compared with the commercial biocatalyst (Novozym 435 (CALB), Lipozyme RM IM and Lipozyme TL IM). Textural properties, loading capacity, enzyme stability under different conditions, and activity versus different substrates were analyzed. Although all of the supports reversibly immobilized lipases via interfacial activation of lipases versus the hydrophobic surface of the support, some of them permitted a significant improvement in the final biocatalyst compared to the reference support or the commercial preparations. Enzyme specificity depended strongly on the used support (e.g., the new ones gave almost null activity versus p-nitrophenyl butyrate). However, there is not a universal optimal support; the “best” support depends on the enzyme, the parameter studied and the substrate utilized. Nevertheless, under the conditions utilized, the preparations showed a very good performance in a diversity of reactions and permitted their reuse (both the biocatalyst and the supports after eliminating the enzyme by washing the enzyme with triton X-100). These supports will permit enlarging the library of immobilized lipase biocatalyst, being supports useful for aqueous or organic medium.

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)

Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites

Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.

Optimization of the coating of octyl-CALB with ionic polymers to improve stability and decrease enzyme leakage

Abstract Lipase B from Candida antarctica (CALB) immobilized on octyl-agarose (OC) was submitted to coating with polyethylenimine (PEI) and dextran sulfate (DS). Using lowly loaded enzyme preparations, the properties of OC-CALB preparations hardly improved, suggesting too large the distance between enzyme molecules. However, using OC-CALB preparations with maximum loading, CALB stability was greatly improved in different conditions after PEI coating. Moreover, the CALB release from the OC support in the presence of detergents, or during thermal or organic solvent inactivations was greatly reduced after this treatment (PEI plus DS coating). The results pointed that the main positive effect of this coating could be derived from the physical intermolecular crosslinking of the CALB molecules with the polymers that reduce the enzyme desorption from the support. The coating of OC-CALB-PEI with DS only produced a minimal improvement on enzyme performance. Even though the enzyme release was much more difficult after physical crosslinking, all enzyme molecules could be released from the OC support combining an ionic detergent (SDS), high buffer concentration, pH 3 and 45 °C, while using the OC-CALB just 2% SDS at pH 7 and 25 °C was enough to release all enzyme. The support could be reused several cycles. Thus, this strategy permitted to greatly reduce the enzyme desorption during operation and to improve enzyme stability while keeping the enzyme immobilization reversibility.