Exploration of Protease Resources in the Gut of Omnivorous Gryllotalpa orientalis (Orthoptera: Gryllotalpidae)

An insect's gut microbiome is an essential "organ" in their life cycle, playing a crucial role by aiding food digestion and nutrient absorption. This study employed both culture-independent and culture-dependent methods to explore the protease resources present in the gut of the omnivorous insect <i>Gryllotalpa orientalis</i>. The findings revealed that the gut extract of <i>G. orientalis</i> contained a diverse array of proteases, including cysteine proteases, pepsin, serine proteases, and trypsin, as well as some unidentified proteases. Furthermore, the protease gene <i>htpX</i>, derived from gut bacterium <i>Priestia megaterium</i> DX-3, has been cloned and recombinantly expressed. The recombinant DX-3-<i>htpX</i> protease exhibited a 61.9-fold increase in fermentation level compared to the DX-3 protease. This protease was characterized as a neutral, heat-resistant metalloprotease with an M48 peptidase domain, and it was observed that the binding of Ca²⁺ to the recombinant protease resulted in the formation of the largest active pocket. This study provides technical support for further development and utilization of functional protein resources in insect gut.