Enzyme methods immobilized

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

Enzymes are immobilized by a variety of methods. Two general types of immobilization procedures are used. The first-type procedures are based on weak interactions between the support and the enzyme and are classified as physical methods. The second-type procedures rest upon the formation of covalent bonds between the enzyme and the support and are classified as chemical methods. 

As a general rule, the optimal immobilization method is found empirically by a process of trial and error, where the selectivity, activity, and operational stability of the enzyme after immobilization are taken into account. The immobilization process is very sensitive to different parameters and is treated as a kind of art [16]. 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

Manufacturer Trade Name Enzyme Source Immobilization method Typical values of initial space velocities at 60°C (bed volumes per hour)... 

Mosbach K (ed) (1976) Immobilized enzymes methods in enzymology, vol 44. Academic, New York... 

Previous accounts of enzymic methods in organic synthesis include four reviews3-6 and a chapter in a book,7 while immobilization of enzymes is discussed at length in three volumes8 10 of Methods in Enzymology. The older literature may be found in an earlier volume of the same collection.11... 

With the help of this method, His-tagged L-lactate dehydrogenase was immobilized. By pumping pyruvic acid as substrate with NADH as cofactor, it was demonstrated that the enzyme was still active in the microchannel. In this case, cofactor was used up. Srinivasan et al. [433] incorporated PikC hydroxylase from Streptomyces venezuelae into a PDMS-based microfluidic channel with a similar approach. The enzyme was immobilized to Ni-NTA agarose beads with an in situ attachment, following the addition of the beads to the microchannel. This enabled the rapid hydroxylation of the macrolide YC-17 to methymycin and neomethymycin (Scheme 4.104) in about equal amounts with a conversion of >90% at a flow rate of 70nl/min. 

Tischer W, Wedekind F (1999) Immobilized enzymes methods and applications. Top Curr Chem 95-126... 

When a homogeneous catalyst, that is an enzyme, is immobilized there are in principle three different approaches [3]. The enzyme can be adsorbed onto a carrier, it can be encapsulated in the carrier, or it can be covalently attached to the carrier. In an extreme case of covalent attachment the enzyme can be cross-linked, making a carrier uimecessary. The choice of immobiUzation method and carrier can greatly influence the properties of the immobiUzed enzyme, ideally improving them. To date many excellent reviews [5-13] and even comprehensive books [14] on the topic of enzyme immobiUzation have been written. Many of these reviews and books use their own division of approaches they range from three to eight and can also include membrane techniques. In order to ease the comparison with chemical catalysts, a division according to reference [3] was chosen. This chapter aims to demonstrate, with examples from the research performed within the COST D25 Action, the importance of the area under discussion. 

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