Studies on Protopectinase-C Mode of Action: Analysis of the Chemical Structure of the Specific Substrate in Sugar Beet Protopectin and Characterization of the Enzyme Activity

Chemical structures of pectic substances degraded by protopectinase-C (PPase-C) were characterized to identify the releasing mechanism of pectin from sugar beet protopectin by the action of that enzyme. The substrate of PPase-C was a polysaccharide isolated from sugar beet pulp by extraction with NaOH and sequential digestions with rhamnogalacturonase (PPase-T), β-1,4-D-galactanase, and α-L-arabinofuranosidase. The structure of this polysaccharide was analyzed by gas-liquid chromatography (GLC), NMR analysis, and gas chromatography-mass spectrometry (GC-MS), and it was identified as α-1,5-L-arabinan. According to our results, arabinan chains seemed to be connected to rhamnogalacturonan through a chain of β-l,4-D-galactan. PPase-C hydrolyzed both linear α-1,5-L-arabinan and ramified L-arabinan in a random manner, producing L-arabinose. From these results, PPase-C could be classified as arabinan endo-1,5-α-L-arabinase EC 3.2.1.99]. Moreover, PPase-C seemed to split the L-arabinan of the polysaccharides connecting the rhamnogalacturonan to the other constituents of the plant cell wall in sugar beet pulp, releasing water-soluble pectin.