An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60؇C) or pH 6.8 (80؇C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn 2؉ , Co 2؉, or Mg 2؉ for thermal stability (stable for 1 h at 82؇C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80؇C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85؇C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.Xylose isomerase (EC 5.3.1.5) is an intracellular enzyme which catalyzes the reversible isomerization of D-xylose to Dxylulose, which is then channeled into either the pentose phosphate (23,24) or the phosphoketolase (14) pathway. These enzymes are also referred to as glucose isomerases because of their ability to convert D-glucose to D-fructose (20). The second catalytic activity is exploited industrially for the production of high-fructose corn syrup from corn starch. This process involves several separate enzymatic steps, including liquefaction of corn starch by ␣-amylase, saccharification by glucoamylase, and isomerization by glucose isomerase (17). Typically, the pH optima (pH opt s) of commercially available glucose isomerases range from 7.5 to 9.0 (45). This limits the reaction temperature used in the industrial processes to 60ЊC because of the formation of browning products (mannose, psicose, and other acidic com...