Among the soil macrofauna, earthworms comprise an essential group of invertebrates (detritus feeders) maintaining soil quality. To effectively digest soil organic matter, they mostly use different digestive enzymes secreted by the glandular cells of the intestine. The aim of this study was to assess the proteolytic activity in the gut extract of one of the earthworm species, Pheretima posthuma (Annelida: Oligochaeta: Lumbricidae), inhabiting soils in India, with a focus on its potential industrial applications. Zymographic examinations of the earthworm gut proteases using the Gel-X-ray film contact print method displayed the presence of eight protease isoforms. When casein was used as a substrate, the earthworm gut proteases were not only found stable at high temperature (60°C) and active in a wide range of pH but also stable toward different metal ions; however, its activity slightly increased in the presence of Ca2+, whereas it decreased in the presence of Mg2+ ions. Using chemical inhibitors and synthetic substrates, the presence of chymotrypsin and trypsin-like serine proteases was revealed. These robust proteases exhibit significant potential for industrial applications, including waste management, biocatalysis, and biofertilizer development, owing to their efficiency in protein decomposition and soil fertility enhancement. This study emphasizes the value of earthworm-derived enzymes as sustainable resources for industrial biotechnology, contributing to processes like agricultural residue bioconversion and supporting circular bioeconomy initiatives.
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