Discovery of microbial glycoside hydrolases via enrichment and metaproteomics.

The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.