Purification, Characterization, and Production of Two Pectic Transeliminases with Protopectinase Activity from Bacillus subtilis

Abstract

We found two enzymes that solubilize pectin from protopectin, tentatively named protopectinase-N (PPase-N) and protopectinase-R (PPase-R), in a culture filtrate of Bacillus subtilis IFO 3134. These enzymes were purified to homogeneity by hydrophobic, cation exchange and size exclusion chromatographies. The molecular weights of PPase-N and PPase-R were estimated to be 43, 000 and 35, 000, respectively, by SDS-PAGE. Their pIs were 9. 4 and 8. 2, respectively. These enzymes were stable in a wide range of pH and temperature. PPase-N and -R released water-soluble pectin by transeliminative cleavage of protopectin. According to their substrate specificities and modes of action, PPase-N and PPase-R could be classified as endo-pectate transeliminase (pectate lyase ; EC 4. 2. 2. 2) and endo-pectin transeliminase (pectin lyase ; EC 4. 2. 2. 10), respectively. Both enzymes were produced in a simple medium containing defatted soybean flour and phosphates. Production of PPase-N was repressed by addition of glucose while that of PPase-R was enhanced by phosphate.