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

Jose Virgen-Ortíz,Roberto Fernandez-Lafuente,Laura Fernandez-Lopez,Nerea Lopez-Carrobles,Cristina Otero,Beatriz Gorines,Sara Pedrero

doi:10.3390/molecules22010091

Abstract

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.

Highlights

Enzyme immobilization is a requirement in the design of most biocatalysts to solve the problem of enzyme solubility [1]
The results show how the recovery of a fully clean support using OC-CALB-PEI-dextran sulfate (DS) is more analysis of thethan protein that remained attached to using the support after being submitted different washing difficult when using
The coating of lipases immobilized on octyl supports with ion polymers strongly reduces the enzyme release during incubation under drastic conditions

Summary

Introduction

Enzyme immobilization is a requirement in the design of most biocatalysts to solve the problem of enzyme solubility [1]. Many authors have tried to develop strategies that allow improving other enzyme properties during this step. This way, stability, activity, resistance to inhibitors, selectivity, specificity or even purity may be improved if a proper immobilization protocol is applied [2,3,4,5,6,7,8,9,10,11,12,13,14,15]. There is the risk of enzyme desorption during operation This produces apparent inactivation of the biocatalyst by loss of active protein and product contamination by the enzyme. Stabilization achieved via physical immobilization is usually moderate, because the support remains necessarily physically active (inert supports are preferred to prevent enzyme-support undesired interactions) [17], and usually cannot compete with multipoint covalent attachment [16]

Methods

Results

Conclusion