Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

Abstract

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%).

Multi-Combilipases: Co-Immobilizing Lipases with Very Different Stabilities Combining Immobilization via Interfacial Activation and Ion Exchange. The Reuse of the Most Stable Co-Immobilized Enzymes after Inactivation of the Least Stable Ones

“…The coating of this three-component co-immobilized combilipase with PEI has also diverse effects depending on the substrate, in agreement with previous positive effects reported of this treatment for the activity of these immobilized enzymes in octyl agarose [91][92][93]. PEI modification of many immobilized enzymes is a usual tool to improve enzyme performance, as stated in the introduction [83].…”

“…Moreover, it may prevent enzyme release of the just physically adsorbed lipase molecules [86,90]. Finally, it has been successfully employed as an adhesive agent in the production of lipase multilayers [91][92][93].…”

“…In this paper, we have used different lipases to prepare a multi (five lipase component)-co-immobilized biocatalyst using octyl-vinyl sulfone agarose as immobilization support [94] and PEI as adhesive agent between the two enzyme layers (the ones in contact with the support surface, that will be the most stable enzymes, and the ones in contact with the polymer, that will be the least stable enzymes) [82,[91][92][93]. Octyl-vinyl sulfone (octyl-VS) has been used to prevent enzyme release from the support under any kind of operation conditions.…”

Immobilized Biocatalysts of Eversa® Transform 2.0 and Lipase from Thermomyces Lanuginosus: Comparison of Some Properties and Performance in Biodiesel Production

“…In fact, it has been proposed to coat Novozym 435 with PEI with some positive results in the biodiesel production process by these biocatalyst [103,104]. Moreover, this treatment, in some instances, has increased lipase activity [93,[105][106][107]. However, in this case, this treatment promoted a slight decrease in the activity of both immobilized biocatalysts.…”

Effects of Enzyme Loading and Immobilization Conditions on the Catalytic Features of Lipase From Pseudomonas fluorescens Immobilized on Octyl-Agarose Beads

“…As hydrophobic support, octyl-agarose beads have been selected, this support has been used to immobilize PFL in many instances (Sztajer et al, 1991;Bastida et al, 1998;Fernandez-Lorente et al, 2003;Rios et al, 2019a) and agarose beads may have some applied interest (Zucca et al, 2016). PFL immobilized on this support become more stable than the enzyme covalently immobilized via multipoint attachment .…”