Polygalacturonic acid trans-eliminase of Xanthomonas campestris

Abstract

Polygalacturonic acid trans-eliminase from the culture fluid of Xanthomonas campestris was purified 66-fold by acetone precipitation, citrate extraction and chromatography on diethylaminoethyl- and carboxymethyl-cellulose. The optimum pH is 9.5 in glycine-sodium hydroxide buffer. Up to 1mm-calcium chloride brings about a remarkable stimulation of the enzyme activity and, at this concentration, no other cations promote or inhibit enzyme action except Ba(2+) ions, which cause complete inhibition. The enzyme degrades polygalacturonic acid in a random manner; it does not act upon polygalacturonate methyl glycoside, although it can cleave partially (68%) esterified pectin. The end products from polygalacturonic acid at 46% breakdown are unsaturated di- and tri-galacturonic acids, in addition to saturated mono-, di- and tri-galacturonic acids. Pentagalacturonic acid is split preferentially into saturated dimer plus unsaturated trimer, or into saturated trimer plus unsaturated dimer; at a lower rate, it is also split into monomer and unsaturated tetramer. Unsaturated pentamer is split into unsaturated dimer plus unsaturated trimer. Tetragalacturonic acid is split some-what preferentially at the central bond to form dimer and unsaturated dimer, but it is also split into monomer and unsaturated trimer. Unsaturated tetramer is split only at the central bond to yield only unsaturated dimer. Trigalacturonic acid is split into monomer and unsaturated dimer. Unsaturated trimer is cleaved into saturated dimer and probably 4-deoxy-l-5-threo-hexoseulose uronic acid, which has not yet been directly identified. Neither saturated nor unsaturated digalacturonic acid is attacked. The unsaturated digalacturonic acid was isolated and proved to be O-(4-deoxy-beta-l-5-threo-hexopyranos-4-enyluronic acid)-(1-->4)-d-galacturonic acid.