Activation of immobilized lipase in non‐aqueous systems by hydrophobic poly‐DL‐tryptophan tethers

Abstract

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule (“tether”) between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most “long” linkers (e.g. polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-μm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n ≈ 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against pnitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.

Polymer Chemistry Applications of Cyrene and its Derivative Cygnet 0.0 as Safer Replacements for Polar Aprotic Solvents

“…The synthetic enzymatic activity was assayed using the propyl laurate assay as previously reported by Schilke and Kelly. [101] …”

Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B

“…Fernandez-Lafuente further reported the immobilization that involves the open form of the lipase (Manoel et al 2015). Schilke and Kelly presented a method for covalent immobilization of Candida antarctica lipase B (CALB) on solid surfaces using a long hydrophobic polytryptophan tether, which resulted in 35 times greater esterification and five times greater hydrolytic activity against p -nitrophenol palmitate (Schilke and Kelly 2008). In addition, Chen et al showed that a small change in the hydrophobic/hydrophilic balance of the epoxy-activated polymer films not only changed the binding ability between polymer films and CALB, but also significantly affected the specific activity of the bound CALB (Chen et al 2008).…”

Application of Hierarchically Porous Chitosan Monolith for Enzyme Immobilization

“…The enzymatic activities of free and immobilized lipases were assessed via the hydrolysis of 4-nitrophenyl palmitate ( p -NPP) (Figure S3b). , First, a 15 mM solution of p -NPP and isopropanol was mixed with a 10 mM phosphate buffer (pH = 7.2) at a ratio of 3.5/96.5 ( p -NPP/phosphate buffer). Owing to the low solubility of p -NPP in the phosphate buffer, a higher concentration of p -NPP may result in precipitation.…”