Our group has developed a functional metaproteomic method, in which we combine cultivation-independent -omics methodologies with the immediacy of activity screening. It has the potential to discover all enzymes with a given activity in an environmental community and does not require prior assumptions about the biocatalyst"s structure. It combines metaproteomic extraction of samples with direct, zymography based, activity screening. The type of zymogram can be easily adapted, to find a variety of desired enzyme functions. In this project we will apply this method to discover novel glycosidases, specifically amylases, cellulases and hemicellulases, as these enzymes are key to a sustainable utilization of renewable plant-based resources. We established two dye-based methods for activity detection. One based on the reaction of iodine with starch (for amylases), the other based on congo red, which can form a complex with cellulose and hemicellulose. For esterases, we used 4-Methylumbelliferone (MU) as a fluorescent substrate. MU can also be tailored to form ester bonds with a desired substrate. We used enantiomeric forms of amino acids to find enantioselective esterases. We performed assays to determine characteristics of this enzyme, like kinetics and E-value. For these assays, we will utilize derivates of methylumbelliferone, with which we already have experience. We found four candidates for different amino acids and varying degrees of enantioselectivity. The highest E-value was with around 1000 a remarkable find.

Taken together, we can screen multiple enzyme classes in environments that naturally select for desired enzyme properties, making them accessible for industrial or medical applications. Our method is versatile, as it can be adapted quickly to not only screen for different properties, but different enzyme classes entirely.