Exploring Active Enzymes Through Functional Metagenomics: Fast Screening of Environmental Samples

Functional metagenomics has emerged as an effective tool for discovering novel enzymes directly from environmental samples, overcoming the limitations of traditional culture-based methods. In this study, we used a functional metagenomic approach on a stool sample from Axis kuhlii , an endemic deer species from Indonesia, to identify active cellulases. We created an efficient workflow for enzyme expression directly in Komagatella phaffii by combining metagenomic sequencing to investigate enzyme diversity, multiplex PCR to build a genes library, and rolling circle amplification (RCA) to streamline the cloning process, eliminating the need for intermediate Escherichia coli transformation and propagation steps. Furthermore, a semi-high-throughput screening method was used to evaluate multiple samples at once, allowing for the rapid identification of active enzymes. Using this approach, we discovered five novel endoglucanases and three β-glucosidases with confirmed enzyme activity. This study shows that functional metagenomics can bridge the gap between computational predictions and experimental validation, providing a reliable platform for enzyme discovery and characterization from complex environmental microbiomes.