CLONING, SEQUENCING AND BIOCHEMICAL-CHARACTERIZATION OF XYLOSE ISOMERASE FROM THERMOANAEROBACTERIUM-SACCHAROLYTICUM STRAIN B6A-RI

The xylose isomerase gene from Thermoanaerobacterium saccharolyticum s train B6A-RI was cloned by complementation using Escherichia coli xyl- 5 mutant strain HB101. One positive clone was detected and the recombi nant plasmid, pZXI6, was isolated. The clone contained the vector pUC1 8 and an insert fragment of 4.5 kb. The cloned xylose isomerase gene ( xylA) was expressed constitutively in E. coli. The gene contained one open reading frame (ORF) of 1317 bp, which corresponds to 439 amino ac id residues. The molecular mass of the gene product was calculated to be 50474 Da from the deduced amino acid sequence. A putative promoter region (Pribnow box), TATAATATATAAT, which repeated twice at the -10 r egion in E. coli, was found 25 bp upstream of the ribosomal binding si te. The deduced amino acid sequence of T. saccharolyticum strain B6A-R I xylose isomerase exhibited very high homology to those from Thermoan aerobacterium thermosulfurigenes 4B (formerly Clostridium thermosulfur ogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridiu m thermohydrosulfuricum 39E). Codon usage in xynA, xynB and xylA showe d a clear propensity for AT-containing isocodons. The native molecular mass of the purified recombinant thermostable xylose isomerase was 20 0 kDa, and the enzyme was a tetramer comprised of identical subunits. The apparent temperature and pH optima for activity of the cloned xylo se isomerase were 80-degrees-C and 7.0 to 7.5, respectively.