Colloid-based multiplexed screening for plant biomass-degrading glycoside hydrolase activities in microbial communities

The enzymatic hydrolysis of polysaccharides into fermentable sugars is a crucial step in the conversion of biomass to lignocellulosic biofuels. An efficient hydrolysis is highly dependent on the identification and characterization of optimal glycoside hydrolases. However, existing techniques for characterizing activity are limited by the range of reaction conditions that can be used, sample complexity, and throughput. The method we present is a multiplexed approach based on nanostructure-initiator mass spectrometry (NIMS) that allows for the rapid analysis of several glycolytic activities in parallel under diverse assay conditions. By forming colloids, it was possible to perform aqueous reactions in tubes and microwell plates despite the substrate analogs' hydrophobic perfluorinated tags. This method was validated by analyzing standard enzymatic parameters (temperature, pH, and kinetics) of [small beta]-glucosidase and [small beta]-xylosidase in separate setups. The multiplexity of this assay system was demonstrated by the simultaneous analysis of [small beta]-glucosidase and [small beta]-xylosidase activities, which was then used to profile environmental samples. Enzymes secreted by microbial communities within these samples were extracted by washing the sample with buffer. Enzymatic activities were directly detected within this crude extract without any further sample pretreatment steps. The multiplexed analysis of [small beta]-glucosidase, exo-/endoglucanase, and xylanase activities was applied for a detailed characterization of previously unknown glycoside hydrolase activities. The results show the suitability of the described method for the rapid screening of crude environmental samples under a wide range of conditions to determine enzyme activities from the microbial communities present within these samples.