The contribution of endogenous cellulase to the cellulose digestion in the gut of earthworm ( Pheretima hilgendorfi: Megascolecidae)

Abstract

Cellulase activity has been detected in the digestive tract of earthworms. However, it has not been well clarified whether the origin of those cellulases are the earthworm themselves or the symbionts. In our study, zymogram analysis suggests that one cellulase (endo-β-1,4-glucanase, EC3.2.1.4) mainly works to digest cellulose in Pheretima hilgendorfi. To identify the cellulase in P. hilgendorfi, we carried out cDNA cloning of the cellulase gene from the digestive tract. A novel cellulase gene was identified from the gut of earthworm. The cDNA encoding cellulase of P. hilgendorfi (phhEG) is 1606 bp with an open reading frame encoding a protein of 449 amino acid residues. The deduced amino acid sequence of P. hilgendorfi cellulase showed higher homology to invertebrate cellulases than bacterium cellulases belonging to the glycosyl hydrolase family (GHF) 9. The phhEG gene was detected in intestinal epithelium cell of midforegut using Northern blot and in situ hybridization. Similarly, specific cellulase activity against carboxymethyl cellulose (CMC) was significantly higher in midforegut tissue. Recombinant phhEG produced by wheat germ cell-free protein synthesis system had a cellulase activity which degrade CMC. In zymogram analysis, the molecular size of cellulase was detected as a single band of 51 kDa from the whole gut contents extracts of P. hilgendorfi, and was very similar to the predicted molecular size of the mature phhEG protein. These results strongly suggested that the earthworm has the capacity to produce the endogenous and functional cellulase around the midforegut, and use this cellulase for their cellulose digestion with the support of intestinal caecum.