Microbial oxidative enzymes are of great interest to the chemical industry because they can potentially replace chemical methods for introducing oxygen into natural and synthetic chemicals. They are also useful as biosensors, as remediants of environmental pollutants, mediators in polymer synthesis, degradation of coal and breakdown of woody pulps for paper production. Generally, the advantage over using enzymes instead of chemical components is the increased purity and yield of the desired product. In many applications, the use of enzymes allows the reaction to proceed under milder, more environmentally benign conditions.

However, because industrial applications are generally run under conditions which are incompatible with naturally occurring enzymes, they must often be mutated to improve their performance. Directed evolution provides the fastest and most effective means of attaining this goal. The crucial steps are in mutagenesis and protein screening (e.g. phage display). A few examples of applications of applications of directed evolution to oxidative enzymes are presented:


The idea behind the engineering effort of peroxidase was to design an enzyme that could readily transfer dyes in the highly alkaline and oxidative conditions of laundry wash water. Peroxidase contains a heme cofactor and is able to oxidize hyroxylated aromatics via cyclic oxidation-reduction:
	1)  CiP + H2O2 --> Cpd I + H2O
	2)  Cpd I + aromatic(red) --> Cpd II + aromatic(ox)
	3)  Cpd II + aromatic(red) --> CiP + aromatic (ox)
Here, the Cpd is the target compound. CiP is peroxidase from the Coprinus cinereus mushroom. The aromatic turns green upon oxidation, thereby providing a colorometric screen for the effectiveness of the reaction.

First, starting with the crystal structure, three mutations were identified and implemented, giving a protein with five fold higher stability than the wild type in oxidative conditions, and 154 fold more stable at the higher temperatures. Error prone PCR was then used to build additional thermostable and peroxide stable mutations. The reaction was run at 50C at varying levels of H2O2. However, it was found that the increased stability was achieved at the cost of the overall activity of the enzyme. In order to compensate for this, a genetic recombination system was implemented, allowing for DNA shuffling with high activity mutants. The final protein had adequate activity and was 174 times more thermally stable and 100 times more peroxide stable than the wild-type enzyme.


Dioxygenases perform a key step in the natural degradation of aromatic compounds by introducing two oxygen atoms across a double bond, breaking the aromatic ring. A directed evolution project, aimed at biphenyl dioxygenase (BP Dox), tried to improve BP Dox ability to degrade polychlorinated biphenyls. BP Dox actually consists of two proteins, BphA1 and BphA2 which exist as a heterohexamer. Starting with BphA1's from different sources with slightly different substrate specificities, a number of new proteins were engineered which could modify polychlorinated biphenyls. Additionally, some of the protein variants could actually attack single ring aromatics such as toluene and benzene. Through recombination, the engineered proteins were able to acquire new functions that were very different from the parent genes.