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.
Enzymatic catalysis to produce molecules such as perfumes, flavors, and fragrances has the advantage of allowing the products to be labeled "natural" for marketing in the U.S., in addition to the exquisite selectivity and stereoselectivity of enzymes that can be an advantage over chemical catalysis. Enzymatic catalysis in organic solvents is attractive if solubility issues of reactants or products, or thermodynamic issues (water as a product in esterification) complicate or prevent aqueous enzymatic catalysis. Immobilization of the enzyme on a solid support can address the generally poor solubility of enzymes in most solvents.We have recently reported on a novel immobilization method for Candida antarctica Lipase B on fumed silica to improve the enzymatic activity in hexane. This research is extended here to study the enantioselective transesterification of (RS)-1-phenylethanol with vinyl acetate. The maximum catalytic activity for this preparation exceeded the activity (on an equal enzyme amount basis) of the commercial Novozyme 435® significantly. The steady-state conversion for (R)-1-phenylethanol was about 75% as confirmed via forward and reverse reaction. The catalytic activity steeply increases with increasing nominal surface coverage of the support until a maximum is reached at a nominal surface coverage of 230%. We hypothesize that the physical state of the enzyme molecules at a low surface coverage is dominated in this case by detrimental strong enzyme-substrate interactions. Enzyme-enzyme interactions may stabilize the active form of the enzyme as surface coverage increases while diffusion limitations reduce the apparent catalytic performance again at multi-layer coverage. The temperature-, solvent-, and long-term stability for CALB/fumed silica preparations showed that these preparations can tolerate temperatures up to 70°C, continuous exposure to solvents, and long term storage.2
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