title: Biochemical Characterization, Thermal Stability, and Partial Sequence of a Novel Exo-Polygalacturonase from the Thermophilic FungusRhizomucor pusillusA13.36 Obtained by Submerged Cultivation.
abstract: This work reports the production of an exo-polygalacturonase (exo-PG) byRhizomucor pusillusA13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%.Ea(d)for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d)values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG fromR. pusilluswith an exo-pectinase fromAspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.
authors.affiliation: Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: Lucas Vinícius
authors.initials: LV
authors.lastname: Trindade
authors.affiliation: Universidade de São Paulo (USP), FFCLRP, Ribeirão Preto, SP, Brazil.
authors.forename: Carla
authors.initials: C
authors.lastname: Desagiacomo
authors.affiliation: Universidade de São Paulo (USP), FFCLRP, Ribeirão Preto, SP, Brazil.
authors.forename: Maria de Lourdes Teixeira de Moraes
authors.identifier: 0000-0002-5026-6363
authors.initials: ML
authors.lastname: Polizeli
authors.affiliation: Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil.
authors.forename: André Ricardo de Lima
authors.initials: AR
authors.lastname: Damasio
authors.affiliation: Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: Aline Margarete Furuyama
authors.initials: AM
authors.lastname: Lima
authors.affiliation: Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: Eleni
authors.initials: E
authors.lastname: Gomes
authors.affiliation: Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista (UNESP), São José do Rio Preto, SP, Brazil.
authors.forename: Gustavo Orlando
authors.identifier: 0000-0001-9902-2557
authors.initials: GO
authors.lastname: Bonilla-Rodriguez
mesh_terms: D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D011096:Polygalacturonase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020103:Rhizomucor / Q000201:enzymology / Q000235:genetics / Q000254:growth & development
chemical_list: D005656:Fungal Proteins; D011096:Polygalacturonase