Purification and properties of a galacturonic acid-releasing exopolygalacturonase from a strain of Bacillus

In esopolygalacturonase [exo-PC;ase; poly (1,4-alpha -D-galacturonide) galacturonohydrolase, EC; 3.2.1.67] was found to be extracellularly produced by Bacillus sp. strain KSM-P443. The exo-PGase was purified to homogeneity, as judged by polacrylamide gel electrophoresis, through sequential column chromatographies. If the enzyme had a molecular weight of approximately 45.000 and an isoelectric point of pH 5.8. The N-terminal sequence was Ser-Met-Gln-Lys-Ile-Lys-Asp-elu-Ile-Len-Lys. Thr-Leo-Eys-Val Phe and had no sequence similarity to those of ether pectinolytic enzymes reported to date. Maximum activity toward polypalacturonic acid (PGA) was observed at 60 degreesC and at pH 7.0 in 100 mM Tris-HCl buffer without requiring any metal ions. When the chain length of aligogalacturonic acids increased, the apparent Km for them decreased, but the h-cat values increased. This is the first bacterial eso-Pease that releases exclusively mono-galacturonic acid from PGA, di-, tri-, tetra-, and penta-galacturonic acids.