HYDROLYSIS OF FRUCTAN IN GRASSES - A BETA-(2-6)-LINKAGE SPECIFIC FRUCTAN-BETA-FRUCTOSIDASE FROM STUBBLE OF LOLIUM-PERENNE

Several different fructan and sucrose hydrolysing enzyme activities we re induced in roots and stubble (mainly leaf sheaths) of Lolium perenn e L. plants after defoliation. Among those activities, a fructan-beta- fructosidase (EC 3.2.1.80) that hydrolyses predominantly beta-(2-6)-fr uctosyl-fructose linkages (6-FEH) was purified from the stubble. The u se of the substrate 6,6-kestotetraose and high-performance anion-excha nge chromatography with pulsed amperometric detection allowed linkage- specific screening and sensitive analysis of enzyme activity. A B-FEH was extensively purified to yield one protein band as revealed by one- dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The 6-FEH was separated from the contaminating beta-(2-1) -linkage-specific fructan-beta-fructosidase and invertase activities ( EC 3.1.2.26) by ammonium sulphate precipitation, lectin-affinity, anio n-exchange and size-exclusion chromatography. The purified 6-FEH was a glycoprotein with an apparent molecular mass of 65 000, as determined by size-exclusion chromatography, and of 69 000 by SDS-PAGE. The 6-FE H had an activity optimum in the range of pH 5.1 to 5.6; Temperatures above 30 degrees C affected the stability of the enzyme activity; howe ver, its temperature stability was increased in the presence of 6,6-ke stotetraose. The purified 6-FEH activity hydrolysed the beta-(2-6)-lin kages in 6,6-kestotetraose and (1&6)-kestotetraose at rates five times faster than the beta-(2-1)-linkages in 1,1-kestotetraose and (1&6)-ke stotetraose. Fructose up to 50 mM did not affect B-FEH activity; conve rsely, sucrose substantially inhibited the enzyme activity. Other disa ccharides did not affect 6-FEH. It is suggested that sucrose might mod ulate 6-FEH activity in vivo.