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, pZXI6, 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 50474Da 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 thermosulfrcrigenes 4B (formerly Clostridium thermosulfuvogenes 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 "C and 7.0 to 7.5, respectively.

Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

“…Among them, only xylanases from Ruminococcus flavefaciens (90 kDa) (64), C. thermocellum (92 kDa) (21), and T. saccharolyticum B6A-RI (130 kDa) (30) are larger than 90 kDa. From the results of denaturing and nondenaturing PAGE, as well as the deduced amino acid sequence, the recombinant endoxylanase from strain JW/SL-YS 485 was determined to be a monomer with an approximate molecular mass of 150 kDa.…”

Cloning, DNA sequencing, and expression of the gene encoding Clostridium thermocellum cellulase CelJ, the largest catalytic component of the cellulosome

“…60 amino acids conserved in several surface layer proteins (30) and extracellular enzymes acting on carbohydrate polymers, e.g., a xylanase of C. thermocellum (GenBank accession number M67438), a xylanase of Thermoanaerobacterium saccharolyticum (26), and a pullulanase of Thermoanaerobacterium thermosulfurigenes EM1 (31). SLH domains are present in OlpA (outer layer protein) (10), OlpB (29), and XynX (GenBank accession number M67438) from C. thermocellum, and those of OlpB have proved to have the ability to bind noncovalently to the peptidoglycan of this bacterium (29).…”

Isolation of a new Thermoanaerobacterium thermosaccharolyticum strain (FH1) producing a thermostable dextranase.

“…The phenotypic characteristics of isolate FH1 were also consistent with those of the Thermoanaerobacterium species, though none of the latter has been reported to produce dextranase. Members of this genus, however, are known for their ability to produce exoenzymes such as amylases, xylanases, and cellulases, which are active at temperatures above 65°C (Ganghofer et al, 1998;Lee et al, 1993;Liu et al, 1996;Shao and Wiegel, 1995;Spreinat and Antranikian, 1992). The T. thermosaccharolyticum type strain DSM571 showed no growth with dextran as the sole carbon and energy source under the employed conditions (Table 1).…”