Cloning and Functional Characterization of an Endoglucanase Gene, cel A From Clostridium Chartatabidum

Abstract

An endoglucanase gene (celA) was isolated from a genomic library of the ruminal bacterium Clostridium chartatabidum. DNA sequence analysis of celA revealed 3 open reading frames (ORFs). ORF 1 and ORF 3 showed homology with xylanase II and xylanase, xynZ from Thermomyces lanuginosus and Clostridium thermocellum, respectively. ORF 2 showed homology with the catalytic domains of endoglucanase, celA (family A) of Clostridium cellulolyticum and endoglucanase, celD of Clostridium thermocellum. Endoglucanase gene, celA of Clostridium chartatabidum was successfully expressed in E. coli and cell extracts of E. coli cells harboring the recombinant plasmid, pcel 1 exhibited both endoglucanase and xylanase activities. Endoglucanase and xylanase activities were optimum at pH 6.0 and 7.2, respectively. Specific endoglucanase and endoxylanase activities were 8.3 and 5.9 μmoles of glucose or xylose equivalent / mg, respectively. Deletion analysis showed that the catalytic domain of endoglucanase gene, celA was located on the HincII (1.1kb) fragment of clone pCel 1. A pUC19 subclone containing the HincII (1.1 kb) fragment was successfully expressed in E.coli, and the levels of its endoglucanase and xylanase activities were equal to those of the entire pcel 1 clone. Xylanase activity was located on the HindIII-HincII fragment of pcel 1. These results show that celA gene encodes a bifunctional enzyme with separate endoglucanase and xylanase domains