title: Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties.
abstract: An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.
authors.affiliation: Laboratório de Enzimologia, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, CEP 70910-900, Brazil.
authors.forename: S Maria C
authors.initials: SM
authors.lastname: Celestino
authors.forename: S
authors.initials: S
authors.lastname: Maria de Freitas
authors.forename: F
authors.initials: F
authors.lastname: Javier Medrano
authors.forename: M
authors.initials: M
authors.lastname: Valle de Sousa
authors.forename: E Ximenes Ferreira
authors.initials: EX
authors.lastname: Filho
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D004795:Enzyme Stability; D003904:Mitosporic Fungi / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011096:Polygalacturonase / Q000032:analysis* / Q000737:chemistry*; D013696:Temperature
chemical_list: D011096:Polygalacturonase