Droplet microfluidics for double lipase immobilisation using TiO2 and alginate microbeads

Abstract

A double immobilisation technique was developed for C. antarctica lipase (CALB) that improved its enzymatic activity and solved difficulties frequently observed in reactions catalysed by lipases. The first immobilisation consisted of CALB adsorption onto a TiO2 nanoparticle surface (CALB TiO2). The adsorption was carried out by an oriented monolayer formed by CALB’s hydrophilic amino acids and the TiO2 surface, leaving the CALB’s active site accessible for reaction. As a result, an increase in enzymatic activity was achieved. The Relative Enzymatic Activity ((REA) obtained was 289%. The second immobilisation consisted of CALBTiO2 entrapment into calcium alginate microbeads [(CALBTiO2)EDTA-Ca], obtained by an internal crosslinking ion-exchange mechanism and using microfluidic droplet technique. The microbiocatalyst obtained, (CALBTiO2)EDTA-Ca, retained a high enzymatic activity (REAt=0 = 232%) and stability (REAt=30days = 263%) at a size (diameter, Ø=8.9·104 nm) that enabled easier recovery than CALBTiO2 (Ø = 2.3·102 nm) or CALBFree (Ø=5.0 nm), and showed a favourable porosity for diffusion without releasing CALBTiO2. Although the microbeads showed CALBFree leaching, as demonstrated by the loss of REA after a mechanical resistant test, (CALBTiO2)EDTA-Ca maintained an almost constant REA.