Laura Fernandez-Lopez

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.

Reversible Immobilization of Lipases on Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption

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.

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.

Stabilizing effects of cations on lipases depend on the immobilization protocol

The effect of an additive on enzyme stability used to be considered an intrinsic feature of a lipase. However, in this paper we have found that the effect of additive on enzyme stability depends on the immobilization protocol. After assaying the effects of diverse chloride salts with different cations on different lipases activity, no relevant effect was detected. Free enzymes or the covalently immobilized enzymes are not stabilized by these cations for any of the studied lipases. However, Mn2+ and Ca2+ (at a concentration of 5 mM) are able to greatly stabilize the lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present during the inactivation, but only if the enzymes are immobilized on octyl-agarose (stabilization factor ranging from 20 to 50). The effect was only detected when using more than 2.5 mM of the cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action. The stabilization seemed to be based on a specific mechanism, and required the recognition sites to be saturated by the cations. Mg2+ has no effect on enzyme stability for both enzymes, but it is able to suppress the stabilization promoted by the other two cations using CRL; while it has no effect on the cation stabilization when using RML. This is the first report of a cation induced enzyme stabilization effect that depends on the lipase immobilization protocol.

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.

Improved stability of immobilized lipases via modification with polyethylenimine and glutaraldehyde

Phospholipase Lecitase Ultra (LU) and lipase from Thermomyces lanuginosus (TLL) have been immobilized under conditions that favor either enzyme crowding or enzyme dispersion. Highly loaded LU was more stable than low loaded biocatalyst under all studied conditions. Using TLL, the results depended on the inactivation conditions, e.g., crowding was positive at pH 5 and negative at pH 7. Then, all preparations were treated with glutaraldehyde (Glu), polyethyleneimine (PEI) or sequentially with Glu and PEI. These treatments may permit to stabilize the physically immobilized lipases by avoiding enzyme desorption via intermolecular crosslinking. Moreover, immobilizing a second enzyme on the lipase-glutaraldehyde-PEI has been proposed as a strategy without risks of PEI desorption by incubation in high ion strength solutions. The treatments altered the enzyme activity slightly but produced significant enzyme stabilization. This enzyme stabilization was more significant when using the highly loaded preparations, where intermolecular crosslinking was easier to obtain. SDS-PAGE analyses confirmed that crowded enzyme preparations were intermolecular crosslinked using Glu plus PEI, but some molecules still remained non-crosslinked. In general, PEI treatment was the most effective in increasing enzyme stability, while glutaraldehyde had a milder stabilization effect.