Kinetic characterization of d-xylose isomerases by enzymatic assays using d-sorbitol dehydrogenase

Abstract

The enzymatic and coupled d-xylose isomerase/ d-sorbitol dehydrogenase assay is a rapid and specific method, permitting accurate quantification of d-xylose isomerization and of d-xylose. The method is based on the isomerization of d-xylose to d-xylulose, followed by reduction of the latter to xylitol by commercially available d-sorbitol dehydrogenase and NADH. The application of this one-step method cannot be extended to d-glucose isomerization since the conditions for a valid coupled assay are not fulfilled. For quantification of d-glucose isomerization, the two-step procedure with d-sorbitol dehydrogenase is recommended. Kinetic parameters for d-xylose and d-glucose using d-xylose isomerase from Streptomyces violaceoruber are reported. The results are compared with the widely used colorimetric cysteine-carbazole method.