Cloning, sequencing and biochemical characterization of xylose isomerase from Thermoanaerobacterium saccharolyticum strain B6A-RI

Abstract

The xylose isomerase gene from Thermoanaerobacterium saccharolyticum strain B6A-RI was cloned by complementation using Escherichia coli xyl-5 mutant strain HB101. One positive clone was detected and the recombinant plasmid, pZX16, was isolated. The clone contained the vector pUC18 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 acid 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 region in E. coli, was found 25 bp upstream of the ribosomal binding site. The deduced amino acid sequence of T. saccharolyticum strain B6A-RI xylose isomerase exhibited very high homology to those from Thermoanaerobacterium thermosulfurigenes 4B (formerly Clostridium thermosulfurogenes 4B) and Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). Codon usage in xynA, xynB and xylA showed a clear propensity for AT-containing isocodons. The native molecular mass of the purified recombinant thermostable xylose isomerase was 200 kDa, and the enzyme was a tetramer comprised of identical subunits. The apparent temperature and pH optima for activity of the cloned xylose isomerase were 80 degrees C and 7.0 to 7.5, respectively.