Immobilization of Enzymes on Fumed Silica Nanoparticles for Applications in Nonaqueous Media

Abstract

Enzymatic catalysis in nonaqueous media is considered as an attractive tool for the preparation of a variety of organic compounds of commercial interest. This approach is advantageous for numerous reasons including the enhanced stability of some substrates and products in solvents, sometimes improved selectivity of the enzyme, and reduction of unwanted water-dependent side reactions since little water is present. Due to the poor solubility of enzymes in these media, mass transfer limitations are sometimes present, leading to low apparent catalytic activity. Immobilization on solid supports has been successfully applied to overcome enzyme solubility issues by increasing the accessibility of substrates to the enzymes' active sites. We have developed a simple immobilization protocol that uses fumed silica as support. Fumed silica is an inexpensive nanostructured material with unique properties including large surface area and exceptional adsorptive affinity for organic macromolecules. Our protocol is performed in two main steps. First, the enzyme molecules are physically adsorbed on the surface of the non-porous fumed silica nanoparticles with the participation of silanol groups (Si-OH) and second, water is removed by lyophilization. The protocol has been successfully applied to both s. Carlsberg and Candida antarctica lipase B (CALB). The resulting fumed silica-based nanobiocatalysts of these two enzymes were tested for catalytic activity in hexane. The transesterification of N-acetyl-L: -phenylalanine ethyl ester was the model reaction for s. Carlsberg nanobiocatalysts. The simple esterification of geraniol and the enantioselective transesterification of (RS)-1-phenylethanol were the model reactions for CALB nanobiocatalysts. The observed catalytic activities were remarkably high and even exceeded those of commercially available preparations.

Investigations on diffusion limitations of biocatalyzed reactions in amphiphilic polymer conetworks in organic solvents

“…It shows a carrier activity of 9.6 U/g in the substrate mixture. Taking into account that the RmL-loading of the Immobead 150 is between 5 and 10 wt.%, the RmLloaded PHEA-l-PEtOx microparticles show nearly tenfold higher carrier activity with some 25-50-fold lower enzyme Cruz et al (2011) describes the lipase immobilization onto fumed silica particles, where the catalytic activity in hexane was just twofold higher than commercially available Novozyme 435. In a work of Bayramoglu et al (2005) for example the immobilization of Candida rugosa lipase on microspheres containing epoxy groups with spacer arms, a 1.5-fold activation could be obtained.…”

Development of protease nanobiocatalysts and their application in hydrolysis of sunflower meal protein isolate

“…The excessive protein loading achieved on CCAFNS without hydrolytic activity could be ascribed to the fact that due to high surface packing density, the distortion of active enzyme conformation is probably promoted by the formation of enzyme‐enzyme interactions upon the attachment on the support surface (Cruz et al ., 2010). Additionally, enzymes tend to maximise contact with the surface of the carrier, which can also alter their native conformation thus leading to reduced activity (Cruz et al ., 2011). These hypotheses are confirmed by very small specific activity (0.1 IU per mg of bound proteins) and activity immobilisation yield (0.98%) of Alcalase ® immobilised on CCAFNS.…”

Recent Developments in Carriers and Non-Aqueous Solvents for Enzyme Immobilization

“…In recent studies, the small pores of the silica SBA (Santa Barbara Amorphous)-15 have been increased from approximately 5 to 30 nm in diameter [26], the volume of the mesopores has been reported to be approximately 0.8 cm 3 /g, and the surface area has been increased up to 1400 m 2 /g [27]. In silica gels, various pore sizes from 70 nm to 250 nm have been obtained [28,29]. Additionally, a non-ordered mesoporous silica material, Hiroshima Mesoporous Material, with a large pore size and no regular shape was prepared in a water/oil phase through organic templates [30,31].…”