(263c) P450 Biocatalysis in Two-Phase Aqueous-Organic Emulsions

Authors: 
Ryan, J., UC Berkeley
Clark, D. S., UC Berkeley


Cytochrome P450s catalyze the oxidation of many diverse compounds that are potentially useful as intermediates for industrial and pharmaceutical syntheses. P450 biocatalysis is an attractive alternative to traditional chemical synthesis as a means to achieve asymmetric hydroxylations, but there are many factors limiting the large-scale integration of P450s into synthetic schemes. For non-natural substrates in particular, inefficient cofactor utilization coupled with poor water solubility can translate to slow reaction rates and low product yields.

As an alternative to aqueous P450 catalysis, two-phase emulsions can harbor the enzyme in an aqueous environment while the organic phase acts as a product sink and a substrate reservoir, increasing the amount of substrate available to the enzyme. In this work, we have shown that two-phase emulsions containing small amounts (<5 wt%) of bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) enable much higher substrate and product concentrations, faster reaction rates, and higher overall product yields for hydroxylation reactions catalyzed by the three-component P450cam from Pseudomonas putida.

Initial kinetic studies were performed with camphor as a model substrate to optimize both the amount of surfactant and buffer content of the emulsions. The decoupling of NADH oxidation relative to substrate turnover was shown to improve with increasing amounts of AOT. The emulsion conditions optimized for camphor hydroxylation by P450cam can also be used for the hydroxylation of many other substrates by both wild-type and mutant forms of P450cam as well as other water soluble P450s. Examples of such reaction systems will be presented. Alternate electron sources can also be used, making the use of a two-phase emulsion more amenable to scale up.