Multidomain and multifunctional glycosyl hydrolases from the extreme thermophile Caldicellulosiruptor isolate Tok7B.1

DNA sequencing techniques have revealed widespread molecular diversity of t he genomic organization of apparently closely related bacteria las judged f rom SSU rDNA sequence similarity). We have previously described the extreme thermophile Caldicellulosiruptor saccharolyticus, which is unusual in poss essing multi-catalytic, multidomain arrangements for the majority of its gl ycosyl hydrolases, We report here the sequencing of three gene clusters of glycosyl hydrolases from Caldicellulosiruptor sp. strain Tok7B.1. These clu sters are not closely linked, and each is different in its organization fro m any described for Cs. saccharolyticus, The catalytic domains of the enzym es belong to glycosyl hydrolase families 5, 9, 10, 43, 44, and 48. The cell ulose binding domains (CBDs) of these enzymes from Caldicellulosiruptor sp. Tok7B.1 are types mb, IIlc, or VI. A number of individual catalytic and bi nding domains have been expressed in Escherichia coli, and biochemical data are reported on the purified enzymes for cellulose degradation encoded by engineered derivatives of celB and celE.