PURIFICATION AND CHARACTERIZATION OF THE FAMILY-J CATALYTIC DOMAIN DERIVED FROM THE CLOSTRIDIUM-THERMOCELLUM ENDOGLUCANASE CELJ

The Clostridium thermocellum endoglucanase CelJ contains two different catalytic domains in a polypeptide, i,e., a subfamily E1 catalytic do main and a family J catalytic domain [J. Bacteriol., 178, 5732-5740 (1 996)]. The family J catalytic domain (CDJ-CelJ) was produced by a reco mbinant Escherichia coli and purified, The purified CDJ-CelJ gave a si ngle band on SDS-polyacrylamide gel electrophoresis and the molecular weight of this enzyme (60,000) was consistent with the value (60,333) calculated from the DNA sequence, CDJ-CelJ hydrolyzed various cellulos ic substrates, xylan, and lichenan but not p-nitrophenyl (PNP)-cellobi oside, PNP-glucoside, or PNP-xyloside at all, CDJ-CelJ was active on A vicel, a microcrystalline cellulose, and the specific activity of CDJ- CelJ on Avicel (0,0078U/mg protein) was comparable to that of CelS, wh ich is recognized as the most important catalytic subunit of the C, th ermocellum, cellulosome, suggesting that CelJ is also an important cat alytic subunit in the cellulosome of this bacterium, in addition to Ce lS.