Preparation of reusable bioreactors using reversible immobilization of enzyme on monolithic porous polymer support with attached gold nanoparticles

Abstract

Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km = 10.9 mmol/L, and maximum reaction rate, Vmax = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel.

Immobilization of pectinase on reusable polymer support for clarification of apple juice

“…In the first approach, enzymes with non-essential -SH groups are immobilized by formation of disulfide (S-S) bonds with a thiol reactive support. The bound protein can be released by treatment with an excess of dithiothreitol[23,24]. Although elegant, it would be difficult to employ this technique on industrial scale.Guisan's group has reported excellent and generally applicable methodologies of enzyme immobilization on reusable supports during last several years[25][26][27].…”

Enzyme immobilization on polymethacrylate-based monolith fabricated via thermally induced phase separation

“…Zhang et al reported that HRP was physically immobilized on graphene oxide (GO) by adsorption due to electrostatic interaction [45]. However, physical attachment may not be strong enough to prevent the loss of enzymes during the operation, which limits wide applications of the immobilized HRP [46]. Hence, covalent binding has attracted more and more attention.…”

Recent developments in biocatalysis beyond the laboratory

“…Another recent development in lipase immobilization is the preparation of a reusable monolithic bioreactor using the reversible immobilization technique (Lv et al 2014). This system is comprised of porous polymer monoliths with thiol functionalities holding gold nanoparticles on which porcine lipase was immobilized.…”