Production, nonaqueous enzyme systems

It is generally held that an aqueous environment is desirable, although not a prerequisite, for the optimal activity of enzymes. It is assumed that in most cases, placing an enzyme in an organic medium will cause deleterious effects to the tertiary structure of the protein. However, if this was not the case, there would be potential advantages for enzyme catalysis in an organic medium. It would clearly facilitate conversion of water-insoluble substrates and aid in product recovery. The thermodynamic equilibria of certain reactions are unfavorable in aqueous systems. Enzyme stereoselectivity has been shown to improve in some nonaqueous enzyme transformations (67,68). 

Although the enzyme from Capsicum has both types of activity, prephytoene pyrophosphate is an isolable product in other situations. The conversion of GGPP (9) to prephytoene pyrophosphate (10) has been demonstrated with a soluble tomato plastid enzyme system and chloroplasts isolated from Phaseolus vulgaris by nonaqueous methods (Spurgeon and Porter, 1983). The only cofactor that appears to be required is Mg + or Mn. There is no requirement for a reduced pyridine nucleotide as exists for the formation of squalene. Prephytoene pyrophosphate (10) appears to be the only isolable intermediate involved in the process. This compound has the same absolute stereochemistiy as presqualene pyrophosphate. 

Nonaqueous enzymatic redox reactions have been limitedby stability owing to solvents and highly reactive substrates (H202). Here we have shown evidence of methods to alleviate these concerns for reactions with CPO. In experimental systems, the in situ production of H202 by GOx was shown to function equally well and more reproducibly than added H202. In situ production is experimentally easier and prevents enzyme deactivation owing to high peroxide levels. GOx was more solvent stable than CPO therefore, the GOx system may be useful for this and other redox systems. 

Enzyme activity is markedly influenced by water activity (a ). Lipases from different sources have been shown to have completely different behavior some exhibit high activity at low a.., whereas others show a high activity at intermediate or very high aw (31). The a also affects the apparent and V ax values measured for lipase-catalyzed hydrolysis and esterification reactions (32). Thermodynamic a can be used to define the relationship between water and other components of the reaction system (33). Thermodynamic a of a solution is defined as the water vapor pressure over the solution divided by the water vapor pressure over pure water at a given temperature. The water content of the biocatalyst is more important in dictating enzyme activity than the total water content in the system. Novozym-435 (Novo Nordisk, Franklinton, North Carolina) from Candida antarctica has a water content of 1-2% (w/w) (34). This enzyme preparation has been shown to give high yields of TAG products under several nonaqueous reaction conditions (5, 35, 36). 

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