Coupling with a Nonprotein Auxiliary Agent

1 Chemoenzymatic Cascade Reaction Based on Redox Chromophore 

As a model system, experiments were carried out using (3S)-l,3-dihydroxypentan-2-one 14, which arises from the reaction of propanal with Li-HPA in the presence of E. coli TK. Addition of excess tetrazohum red and sodium hydroxide solution led to a development of a red color within 2 min because of the formation of formazane concomitantly with the diketone. The intensity displayed increased with rising concentrations of 14. According to measurements carried out at 485 nm 14 was detectable down to a corresponding bioconversion of 2.5 mM. As an alternative product from carbohgation to benzaldehyde, l,3-dihydroxy-3-phenylpropan-2-one 15 was also tested in the assay, and comparable levels of detection were determined. 

This colorimetric determination of ketose formation by a redox transformation has several limitations. The method is restricted to nonhydroxylated aldehyde acceptors and requires further handhng steps by addition and removal of solid reagent to ehminate residual Li-HPA. Lastly, this method, based on an endpoint determination of the reaction product, does not allow continuous measurement of enzyme kinetics. 

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