Introduction

Enzymes, i.e. proteins mediating specific, catalytic functions, are amongst the most powerful molecular machines invented by nature. Since decades, humans utilize naturally occurring enzymes as bio detergents (e.g. in washing powder kirk2002industrial), in the paper industry bajpai1999application and for food processing alkorta1998industrial.
The engineering of novel enzymes catalyzing reactions that do not or only inefficiently occur in nature holds great promise for biotechnological production of regenerative fuel, biomaterials and novel pharmaceuticals, e.g. based on Organosilicons. However, so far, enzyme engineering has typically been a time-consuming, elaborate, expensive and inefficient process, usually requiring laborious, iterative trial-and-error optimization of engineered candidates packer2015methods .
To accelerate the development of novel enzymes, our team harnessed the engineering strategy nature uses: Evolution.

Background

The human Cytochrome CYP1A2 is an example of a heme-dependent-thiolate monooxygenase, member of the P450 superfamily and plays an important role in the metabolism of many structurally unrelated substrates. Primarily the CYP1A2 is located in the endoplasmic reticulum of liver cells. It is of great interest as it is involved in the oxidative metabolism of many commonly used therapeutics and xenobiotics ahn2004high, such as caffeine. Caffeine undergoes degradation by CYP1A2 through an initial N3- demethylation into three xanthine derivates, 81.5% paraxanthine, 10.8% theobromine and 5.4% theophylline perera2010caffeine. The chemical structure of these three primary metabolites of caffeine only differ in their methylation pattern (Fig.3).