Improving the kinetic properties of dehydrogenases

Because substrate concentrations are normally maintained at high levels to maximize volumetric productivity, turnover number and resistance to product inhibition are critical dehydrogenase properties. Compared with other enzyme classes, many dehydrogenases have relatively low kcat values on the order of 1-10 s-1.35 Increasing the turnover number allows lower catalyst loading in the reactor, which directly translates to lower biocatalyst cost. It also has the indirect benefit of reducing components such as whole cells and proteins that often complicate downstream processing steps by promoting emulsion formation. Directed evolution strategies offer a logical path toward improving turnover number.

Product inhibition is highly undesirable in bioprocesses since it limits the final product titer and volumetric productivity. Two types of product inhibition should be distinguished. Competitive binding of the alcohol product to the dehy-drogenase active site that blocks substrate access is the biochemical definition of product inhibition. Bioprocesses, particularly those utilizing whole cells, may also be subject to a different type of product inhibition more properly referred to as product toxicity. In this case, the alcohol product disrupts a key property of the biocatalyst such as the integrity of the cell membrane or a metabolic process responsible for cofactor regeneration. The distinction is important because classical product inhibition may be addressed by alterations to the dehydrogenase itself; overcoming product toxicity requires a more holistic approach.

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