title: Purification and characterization of an extracellular pectate lyase from an Amycolata sp.
abstract: The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.
mesh_terms: D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D016974:Dickeya chrysanthemi / Q000201:enzymology / Q000235:genetics; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase; C003181:polygalacturonic acid