KINETIC-STUDIES ON P-NITROPHENYL-CELLOBIOSIDE HYDROLYZING XYLANASE FROM CELLVIBRIO-GILVUS

Reactions of the p-nitrophenyl-cellobioside (G2-pNP) hydrolyzing xylanase from Cellvibrio gilvus (XCEL) were investigated kinetically in detail. XCEL hydrolyzed only aglyconic bonds in various arylcellobiosides with close kinetic paramters together. Its kinetic parameters toward various p-nitrophenyl cellooligosaccharides were also close. Two more xylanases, from Streptomyces sp. E86 (XSTR) and from Aspergillus japonicus (XASP), were found to hydrolyze G2-pNP at a lower rate compared with XCEL. The V(max) of XSTR and XASP were comparable to that of XCEL, suggesting that the high G2-pNP-hydrolyzing activity of XCEL was due to its small K(m). A xylanse from Robillarda sp. Y-20 (XROB) did not have any activity on G2-pNP. p-Nitrophenyl-xylobioside (X2-pNP) and p-nitrophenyl-glucosyl-xyloside (GX-pNP) were examined as substrates to the four xylanases. Three of the four xylanases hydrolyzed these substrates, only at their aglyconic bonds, rather faster than xylan, but XROB hydrolyzed them with a very small rate. Classification of xylanases based on their activity on the aryl-glycosides is discussed. The advantage of using X2-pNP or GX-pNP for xylanase assay is also discussed.