The usage of Nature’s catalysts for industrial applications is promising to be one of the central pillars of sustainable European chemistry, due to the reduced environmental impact of ‘green’ biocatalytic processes compared to current hazardous chemical procedures. However, at present a large fraction of chemical transformations cannot be substituted with biotechnological production lines, because suitable biocatalysts satisfying the criteria are not known. Our incomplete understanding of enzyme mechanisms makes it difficult to generate catalysts de novo with protein design techniques. An alternative route is the identification of biocatalysts from the natural collections of evolved proteins that fit into the catalytic task to some extent (a phenomenon known as catalytic promiscuity) and improve their catalytic performance for the desired reaction by directed protein evolution techniques. In order to reach this strategic goal, we aim to employ a new high throughput technology (in vitro compartmentalization in microfluidic water-in-oil emulsion droplets) in the search for new biocatalysts. By this assay technology, previously inaccessibly large enzyme libraries will be screened. After isolation of the target enzymes from microbial metagenomes, the latest protein evolutionary theories in relation to catalytic promiscuity will be investigated on a large set of natural enzyme variants, in order to elucidate how new activities are evolved in nature. The resulting aspects of evolution of new enzyme functions could provide an intellectual framework to rationalise evolutionary developments and to utilize these principles for evolution in the test tube for relevant applications.