Biocatalytic processes are gaining increasing importance in organic synthesis from their unique selectivity advantages over traditional methods.  Over the last two decades, biocatalysis has emerged as an important technology for meeting the demand for green and sustainable synthesis of pharmaceuticals, flavor, fragrances, vitamins and other fine chemicals. Our laboratory has developed hybrid P450 enzymes containing a photosensitizer covalently attached to P450 BM3 heme domains. High photocatalytic activity has recently been achieved with a hybrid mutant enzyme in the hydroxylation of natural substrates such as long chain fatty acids. In an effort to expand the synthetic potential of our light-driven hybrid enzyme, we have sought to improve activity toward non-natural substrates. Rational design has been useful for the development of the light-driven methodology and initial steps toward the oxidation of non-natural substrates. Recently we have made good strides in implementing the directed evolution technology in our laboratory. A breakthrough came with the development of a fast and simple way to purify and covalently attach the photosensitizer to heme domain mutants in a one-step process. In combination with our recently developed class of chromogenic substrates, which have been shown to be compatible with the light-driven process, the throughput screening of a library of mutants generated by error-prone PCR has now been possible. We will present our latest results on some interesting rationally designed mutants as well as our efforts toward the directed evolution of hybrid P450 BM3 enzymes.

Pitting rational against random: Improving light-driven P450 biocatalysts

251st National Meeting (2016)

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