2 EXTREMELY THERMOSTABLE XYLANASES OF THE HYPERTHERMOPHILIC BACTERIUMTHERMOTOGA-MARITIMA MSB8

During growth with xylose or xylan as the source of carbon, xylanase p roduction by Thermotoga maritima MSB8 was enhanced about 10-fold compa red with growth viith glucose or starch. Two extremely thermostable en doxylanases (1,4-beta-D-xylan-xylanohydrolase, EC 3.2.1.8), designated XynA and XynB, were identified and purified from cells of this organi sm, XynA and XynB occurred as proteins with apparent molecular masses of about 120 and 40 kDa, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximum activity at the o ptimal pH (PH 6.2 and pH 5.4 for XynA and XynB, respectively) was meas ured at about 92 degrees C for XynA (10-min assay) and at about 105 de grees C for XynB (5-min assay). XynB activity was stimulated twofold b y the addition of 500 mM NaCl, while XynA displayed maximum activity w ithout the addition of salt. Both xylanases were tolerant of relativel y high salt concentrations. At 2 M (about 12% wt/vol) NaCl, XynA and X ynB retained 49 and 65%, respectively, of their maximum activities. In contrast to XynB, XynA was able to adsorb to microcrystalline cellulo se. Antibodies raised against a recombinant truncated XynA protein cro ss-reacted with XynB, indicating that the enzymes may have sequence or structural similarities. Part of the xylanase activity appeared to be associated with the outer membrane of T. maritima cells, since more t han 40% of the total xylanase activity present in the crude cellular e xtract was found in the membrane fraction after high-speed centrifugat ion. Most of the membrane-bound activity appeared to be due to the 120 -kDa xylanase XynA.