Molecular cloning of glycoside hydrolase family 45 cellulase genes from brackish water clam Corbicula japonica

Abstract

We previously reported endogenous Glycoside Hydrolase Family (GHF) 9 beta-1,4-glucanase gene, CjCel9A, from common Japanese freshwater clam Corbicula japonica. Here we identified another endogenous beta-1,4-glucanase genes which belong to GHF45 (CjCel45A, CjCel45B). Both genes encode ORF of 627 bp corresponding to 208 amino acids. CjCel45A and CjCel45B are different in 5' and 3'-untranslated regions and six nucleotides in the ORF. CjCEL45 has only one GHF45 catalytic domain without any carbohydrate binding modules as is the case with other molluskan GHF45 enzymes. Phylogenetic analysis and genomic structure of CjCel45 gene implies that this gene is likely to be acquired from fungi by common ancestor of mollusks. Reverse transcription (RT)-PCR analysis and in situ hybridization revealed that CjCel45A is likely to be expressed in the secretory cells in the digestive gland, suggesting that this cellulase is produced in the same site as CjCEL9A. CjCEL45A was successfully expressed in E. coli cells and zymographic analysis of the recombinant CjCEL45A showed that CjCEL45A is a functional beta-1,4-glucanase. The finding of multiple cellulase genes in C. japonica strongly supports our hypothesis that this species function as a cellulose decomposer in estuarine environments.