Bisepoxide-activated Hollow Silica Microspheres for Covalent Immobilization of Lipase from Burkholderia cepacia

Abstract

An efficient and easy-to-perform method was developed for covalent immobilization of lipase from Burkholderia cepacia (Lipase PS) on hollow silica microspheres (M540) by bisepoxide activation. For immobilization, various bisepoxides of different length, rigidity and hydrophobicity in their linkers were applied to activate the amino groups on the M540 support. Effect of the individual bisepoxides on the catalytic performance of the immobilized Lipase PS was studied by using lipase-catalyzed kinetic resolution (KR) of racemic 1-phenylethanol (rac-1) with vinyl acetate in batch mode. Catalytic activity, enantiomer selectivity, recyclability and thermal stability of the new immobilized Lipase PS biocatalysts were investigated. The optimal enzyme / support ratio with the support activated by the most efficient bisepoxide, i.e. poly(ethylene glycol) diglycidyl ether (PDE), was 1:5. The most efficient Lipase PS on PDE activated M540 showed an almost five fold higher biocatalytic activity value (rbatch = 42.8 U/g) with enhanced selectivity (ee(R)-2 = 99.1 %) to the free form of Lipase PS (rbatch = 9.0 U/g; ee(R)-2 = 98.9 %). The Lipase PS on PDE-M540 was compared to a commercially available immobilized Lipase PS biocatalyst (Lipobond Lipase PS) and also applied in a packed-bed enzyme reactor operated in continuous-flow mode, where the optimal temperature of M540-PDE-PS reached the 70 °C, while the optimum for Lipobond Lipase PS was 50 °C.

Conservation of the Biocatalytic Activity of Whole Yeast Cells by Supported Sol – Gel Entrapment for Efficient Acyloin Condensation

“…In case of the 2 nd generation sol -gel method, wholecells and a proper inert solid support were co-immobilized. Hollow silica microspheres with amino/vinyl grafted surface (M540) were previously described as efficient enzyme carrier [41,42], thus M540 was tried in 2 nd generation sol -gel systems as support material as well.…”

The rise of continuous flow biocatalysis – fundamentals, very recent developments and future perspectives

“…The introduced bisepoxide moieties can covalently bind amine-, thiol-and carboxylate-functional groups under mild conditions. 149 First results with bisepoxide-functionalized silica microspheres showed a strong tendency to link the enzyme compared to other glutaraldehyde-based support-activated functionalization strategies. 149 Bisepoxides with long and flexible chains can also provide multi-point binding of the enzyme to the carrier to promote an even more robust anchoring.…”

“…149 First results with bisepoxide-functionalized silica microspheres showed a strong tendency to link the enzyme compared to other glutaraldehyde-based support-activated functionalization strategies. 149 Bisepoxides with long and flexible chains can also provide multi-point binding of the enzyme to the carrier to promote an even more robust anchoring. Moreover, the immobilized enzymes preserve their activity and selectivity in contrast to other covalent immobilization strategies because the enzyme's catalytically active site is not sterically hindered, especially if a longer and more flexible bifunctional linker is used.…”

“…A broad variety of materials are used as carriers and they are classified into organic, inorganic, hybrid or composite substances depending on their chemical composition. 149 As aforementioned, covalent attachment is the most commonly applied procedure for attaching an enzyme to a solid support. However, an activation either of the enzyme or the support (or also both) is needed to achieve the required bound formation.…”

“…Several bisepoxides with different lengths, stiffnesses and hydrophobicities were selected and evaluated for the surface modification of hollow silica microspheres. 149,152 The epoxy functions of the activator-agent bind covalently to the enzymes instead of a simple adsorption on the silica surface. The introduced bisepoxide moieties can covalently bind amine-, thiol-and carboxylate-functional groups under mild conditions.…”