Hydrolysis of fructan in grasses: a β-(2-6)-linkage specific fructan-β-fructosidase from stubble of Lolium perenne

Abstract

Several different fructan and sucrose hydrolysing enzyme activities were induced in roots and stubble (mainly leaf sheaths) of Lolium perenne L. plants after defoliation. Among those activities, a fructan-β-fructosidase (EC 3.2.1.80) that hydrolyses predominantly β-(2-6)-fructosyl-fructose linkages (6-FEH) was purified from the stubble. The use of the substrate 6,6-kestotetraose and high-performance anion-exchange chromatography with pulsed amperometric detection allowed linkage-specific screening and sensitive analysis of enzyme activity. A 6-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 β-(2-1)-linkage-specific fructan-β-fructosidase and invertase activities (EC 3.1.2.26) by ammonium sulphate precipitation, lectin-affinity, anion-exchange and size-exclusion chromatography. The purified 6-FEH was a glycoprotein with an apparent molecular mass of 65000, as determined by size-exclusion chromatography, and of 69000 by SDS–PAGE. The 6-FEH had an activity optimum in the range of pH 5·1 to 5·6. Temperatures above 30°C affected the stability of the enzyme activity; however, its temperature stability was increased in the presence of 6,6-kestotetraose. The purified 6-FEH activity hydrolysed the β-(2-6)-linkages in 6,6-kestotetraose and (1&6)-kestotetraose at rates five times faster than the β-(2-1)-linkages in 1,1-kestotetraose and (1&6)-kestotetraose. Fructose up to 50 mM did not affect 6-FEH activity; conversely, sucrose substantially inhibited the enzyme activity. Other disaccharides did not affect 6-FEH. It is suggested that sucrose might modulate 6-FEH activity in vivo.