NUCLEOTIDE-SEQUENCE OF THE CLOSTRIDIUM-STERCORARIUM XYNB GENE ENCODING AN EXTREMELY THERMOSTABLE XYLANASE, AND CHARACTERIZATION OF THE TRANSLATED PRODUCT

The nucleotides of the xynB gene of Clostridium stercovarium F-9 were sequenced. The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377. The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus. Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E. coli and C. stercorarium F-9. The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside. The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C. This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min.