PURIFICATION AND CHARACTERIZATION OF AEROMONAS-CAVIAE ME-1 XYLANASE-V, WHICH PRODUCES EXCLUSIVELY XYLOBIOSE FROM XYLAN

A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the xylanase were 46 kDa and 5.4, respectively. Xylanase V had a maximum activity at a pH of 6.8 and at a temperature between 30 and 37 degrees C. It was relatively stable at a pH between 5.0 and 8.6 and a temperature between 25 and 37 degrees C. When soluble birch xylan was used as the substrate, the enzyme had a K-m, and V-max, of 2 mg/ml and 182 mu mol of xylose equivalent liberated min(-1) mg of protein(-1), respectively. By the action of xylanase V on xylans (from oat spelt and birch), only one product corresponding to xylobiose was observed by thin-layer chromatography. The xylanase V putative product was confirmed to be xylobiose by acid and enzymatic hydrolyses. The xylanase had neither beta-xylosidase, alpha-L-arabinofuranosidase, cellulase, nor beta-1,3-xylanase activities. Xylotriose was the shortest substrate which the enzyme could attack. These findings suggest that xylanase V is a novel enzyme that cleaves a xylobiose unit from one of the ends of xylans, probably by an exomechanism.