Polygalacturonase of Erwinia carotovora.

The polygalacturonase, clearly differentiated from polygalacturonic acid trans-eliminase, produced in the culture fluid of Erwinia carotovora was purified 56-fold by means of acetone precipitation, acetate butfer extraction, and two successive treatments by chromatography on carboxymethyl cellulose. The purified enzyme is specific for nonmethoxylated polygalacturonic acid. It shows an optimum pH between 5.2 and 5.4; no shift of optimum pH is observed when tetragalacturonic acid is used as a substrate. The activity is stimulated by 0.1 M sodium, potassium, and ammonium ions, but not by 0.001 M calcium, magnesium, and barium ions. At 0.1 M, phosphate (pH 5.2), chloride, and sulfate ions are somewhat inhibitory as compared to acetate ions, but 0.1 M phosphate (pH 4.8) and 0.001 M polymetaphosphate ions are not inhibitory. The major end products are monoand digalacturonic acids. Digalacturonic acid is not hydrolyzed further. The relative initial rates of the reactions on substrates of different chain lengths are: polygalacturonic acid, 100; shorter chain polygalacturonic acid (average degree of polymerization, 12), 52; hexagalacturonic acid, 15.8; pentagalacturonic acid, 12.8; tetragalacturonic acid, 2.2 ; trigalacturonic acid, 1.7. Hexamer is cleaved predominantly at the central bond, producing 2 molecules of trimer; slower reactions yielding dimer and tetramer were also observed. Pentamer is cleaved only into dimer and trimer. Tetramer is cleaved at Bond 1 to yield trimer and monomer; the central bond of tetramer is also cleaved at a lower but significant rate to form dimer. Both reduced and oxidized tetramer are cleaved much more slowly, and at the central bond only, resulting in an accumulation of normal and reduced or oxidized dimer. Simultaneous attack of Bonds 1 and 2 is postulated for trimer hydrolysis, since only the monomer is found as the reaction product and no dimer is detected, although Erwinia polygalacturonase cannot hydrolyze the dimer. Both reduced and oxidized trimer are cleaved at Bond 1 after a long incubation.