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 microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities using the carbohydrate polymer pullulan at elevated temperatures and acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6 glycosidic bonds. Along with its hydrolyzing enzymes, it has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and soil from the bank of a pond. In both cases, Alicyclobacillus emerged as the dominant microorganism. Metaproteomic analysis of the enrichment biomass and secretome identified several pullulan-degrading enzymes from this organism. Notably, these enzymes were absent in the metagenomic analysis of the initial inoculum, underscoring the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes from complex microbial environments.