Polymer-enzyme ratio

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]. 

The effects of the feed ratio in the lipase CA-catalyzed polymerization of adipic acid and 1,6-hexanediol were examined by using NMR and MALDI-TOF mass spectroscopies. NMR analysis showed that the hydroxyl terminated product was preferentially formed at the early stage of the polymerization in the stoichiometric substrates. As the reaction proceeded, the carboxyl-terminated product was mainly formed. Even in the use of an excess of the dicarboxylic acid monomer, the hydroxy-terminated polymer was predominantly formed at the early reaction stage, which is a specific polymerization behavior due to the unique enzyme catalysis. 

The peroxidase-catalyzed polymerization of m-alkyl substituted phenols in aqueous methanol produced soluble phenolic polymers. The mixed ratio of buffer and methanol greatly affected the yields and the molecular weight of the polymer. The enzyme source greatly affected the polymerization pattern of m-substituted monomers. Using SBP catalyst, the polymer yield increased as a function of the bulkiness of the substituent, whereas the opposite tendency was observed when HRP was the catalyst. 

The concentration of the synthase or the number of enzyme copies has been assumed to have an influence on the molecular mass and molecular mass distribution of the synthesized polymer [33],but this has not been confirmed. The only variables found so far to control the molecular mass of the polymer are the initial ratio of substrate to enzyme levels, and the concentration of inducing factors in the culture medium [34-36] cf. also Chap. 9 of this book. 

The major part of the reports discussed above provides only a qualitative description of the catalytic response, but the LbL method provides a unique opportunity to quantify this response in terms of enzyme kinetics and electron-hopping diffusion models. For example, Hodak et al. [77[ demonstrated that only a fraction of the enzymes are wired by the polymer. A study comprising films with only one GOx and one PAH-Os layer assembled in different order on cysteamine, MPS and MPS/PAH substrates [184[ has shown a maximum fraction of wired enzymes of 30% for the maximum ratio of mediator-to-enzyme, [Os[/[GOx[ fs 100, while the bimolecular FADH2 oxidation rate constant remained almost the same, about 5-8 x 10 s ... 

In a like manner, a co-polymer of styrene and acryloxysuccinimide with a 10 to 1 ratio was prepared. Enzymes immobilized on this type of polymer had different physical properties. They are soluble in organic solvents such as dioxane and DMF, but insoluble in aqueous solutions. Lipases and cholesterol esterase immobilized on this type of polymer are very stable and active in several organic solvents, and have been used in several enantioselective transformations. The protocols for the immobilization are depicted in Figure 13. 

With the increased use of enzymes in polymer chemistry, the enzymology terminology to describe the reaction kinetics and the enantioselectivity of a reaction has become more and more common in polymer literature. The parameter of choice to describe the enantioselectivity of an enzyme-catalyzed kinetic resolution is the enantiomeric ratio E. The enantiomeric ratio is defined as the ratio of the specificity constants for the two enantiomers, R) and S) (1) ... 

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