Partial purification and properties of phleinase induced in stem base of orchardgrass after defoliation.

Phleinase induced in stem base of orchardgrass (Dactylis glomerata L.) after defoliation was partially purified with ammonium sulfate precipitation, DEAE-Sephadex chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The molecular weight of phleinase was 57,000 as determined by gel chromatography. The enzyme showed normal Michaelis-Menten kinetics and its K(m) value was 91 millimolar for phlein of mean degree of polymerization 60 as substrate. Reaction velocity of the enzyme was proportional to molarity of phlein irrespective of its chain length (mean degree of polymerization, 30 to 314). Phleinase attacked terminal fructosyl linkage of phlein by multi-chain mechanism. Phleinase cleaved beta-2,6 linkage, beta-2,6 linkage branched with beta-2,1 linkage, and beta-2,1 linkage of fructan in order of affinity, but not sucrose. Phleinase exhibited an optimum activity at pH 5.5 at 40 degrees C. Its complete inactivation occurred at 60 and 70 degrees C without and with phlein, respectively. Heat inactivation of the enzyme was enhanced by p-chloromercuribenzoate and protected partially by l-cysteine. The enzyme was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and Hg(2+). The modes of action of phleinase were compared with those of the related enzymes.