Priestia megaterium Genome sequencing. Priestia megaterium

The gut microbiota is a vital "organ" of insects, playing an essential role in their life cycle. With the gradual revelation of the biological functions of the gut microbiota, the abundant microbial and digestive enzyme resources in the insect gut urgently need to be developed and utilized. Based on microbial, gene recombination, and methods for combining enzyme inhibitors, zymography, and mass spectrometry, the protease resources in the gut of omnivorous Gryllotalpa orientalis were explored. Four proteolytic bacteria were screened and identified as Priestia aryahattai, P. megaterium, and Serratia surfactantfaciens. The protease gene htpX derived from strain DX-3 was cloned, and a recombinant strain WB800N/pHT43-htpX was constructed. The recombinant DX-3-htpX protease showed a 61.9-fold increase in fermentation level compared to DX-3 protease and was a neutral heat-resistant protease. The 3D structure and binding pocket analysis of the DX-3-htpX protease indicated it was a metalloproteinase containing an M48 peptidase domain. The analysis of the first three binding pockets revealed that the primary amino acid groups at the active center of the protease were methionine, alanine, serine, and threonine. The protease resources in the gut extract of G. orientalis were cysteine proteases, pepsin, serine proteases, and trypsin. The information of 18 proteolytic enzymes derived from the gut was preliminarily identified using protein mass spectrometry, among which 28 and 35 kDa protein bands may contain unrecognized protease. The screening and analysis of proteolytic bacteria and enzymes were conducive to the subsequent development and utilization of the gut-functional microorganisms and genetic resources of insects.