title: Substrate binding ability of chemically inactivated pectinase for the substrate pectic acid.
abstract: Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave Kd values of 5.3 and 8.5 mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.
authors.affiliation: National Food Research Institute, Tsukuba, Ibaraki, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Chiba
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism*; D011096:Polygalacturonase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D011134:Polysaccharides; D010368:Pectins; D011096:Polygalacturonase; C003181:polygalacturonic acid