Directed Evolution of Alkane Degrading Enzymes

Wild type strains screened for hydrocarbon degrading activity.

Reduction of high molecular weight compounds and polymers into finer constituent lengths has pertinence in biochemical upgrading and production of oil from biological waste, environmental applications of degradation in toxic or recalcitrant conditions, and management of bacterial compounds. Many of the most recalcitrant and persistently harmful compounds are of high molecular weight: polymers such as PVC and hydrocarbon fractions such as asphaltenes. While fungi have a function of extracellular enzyme secretion, operable bacteria have limited degradation opportunity with low rates of diffusion across the cell wall and exclusivity to contact with medium size compounds due to light reaction's toxicity and heavy fraction's low water solubility.

The advantages of an enzymatic method above and beyond the application of bacteria have been particularly manifest in respect to flexibility in mixed and hazardous environments – most compounds inhibitory to microbial growth would not affect an enzyme, unless it was an enzyme inhibitor. There are fewer limitations on delivery and it is better suited for continuous flow reactors, among many other industrial applications. In enzymatic reactions there is however a constant need for cofactors, which act as reducing and oxidizing agents. Bacterial enzymes capable of degrading high molecular weight hydrocarbons are furthermore limited to adding onto the terminal or second carbons of alkanes. If these terminal ends are unavailable, further bioremediation is capped, thus enzymes capable of breaking carbon-carbon bonds in the middle of an alkane would affect bioremediation of polymers and compounds such as asphaltenes.

Directed evolution involves introducing mutations into a gene or genes and then looking for improvements over the original or wild type enzyme. Methods of introducing transformations vary widely, but some of the most common include mutator strains, radiation and errorprone Polymerase Chain Reaction (PCR). Because of the complexity of enzyme structure and function, most mutations, even single conservative mutations distant from the active site, can delegate effects from substrate recognition up onto catalysis itself. Therefore we are led to test large numbers of mutants with screening. A typical enzyme of 300 amino acids has a total of 20^300 possible combinations, a surpassing number. Fast methods, typically colorimetric or spectrophotometric, are asked to qualify genetic properties among tens of thousands of variations in an assay.