title: Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii.
abstract: β-Xylosidases are critical for complete degradation of xylan, the second main constituent of plant cell walls. A minor β-xylosidase (BXYL II) from Penicillium janczewskii was purified by ammonium sulfate precipitation (30% saturation) followed by DEAE-Sephadex chromatography in pH 6.5 and elution with KCl. The enzyme presented molecular weight (MW) of 301 kDa estimated by size exclusion chromatography. Optimal activity was observed in pH 3.0 and 70-75 °C, with higher stability in pH 3.0-4.5 and half-lives of 11, 5, and 2 min at 65, 70, and 75 °C, respectively. Inhibition was moderate with Pb+2and citrate and total with Cu+2, Hg+2, and Co+2. Partially purified BXYL II and BXYL I (the main β-xylosidase from this fungus) were individually immobilized and stabilized in glyoxyl agarose gels. At 65 °C, immobilized BXYL I and BXYL II presented half-lives of 4.9 and 23.1 h, respectively, therefore being 12.3-fold and 33-fold more stable than their unipuntual CNBr derivatives (reference mimicking soluble enzyme behaviors). During long-term incubation in pH 5.0 at 50 °C, BXYL I and BXYL II glyoxyl derivatives preserved 85 and 35% activity after 25 and 7 days, respectively. Immobilized BXYL I retained 70% activity after 10 reuse cycles of p-nitrophenyl-β-D-xylopyranoside hydrolysis.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (ICP), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie, 28049, Madrid, Spain. cesarterrasan@hotmail.com.|Departamento de Bioquímica e Microbiologia, Instituto de Biociências, Univ Estadual Paulista (UNESP), Caixa Postal 199, Av. 24 A, 1515, Bela Vista, Rio Claro, SP, 13506-900, Brazil. cesarterrasan@hotmail.com.
authors.forename: César Rafael Fanchini
authors.identifier: http://orcid.org/0000-0002-7988-4755
authors.initials: CRF
authors.lastname: Terrasan
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (ICP), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie, 28049, Madrid, Spain.
authors.forename: Maria
authors.identifier: http://orcid.org/0000-0002-9037-9485
authors.initials: M
authors.lastname: Romero-Fernández
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (ICP), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie, 28049, Madrid, Spain.
authors.forename: Alejandro H
authors.identifier: http://orcid.org/0000-0003-4412-1579
authors.initials: AH
authors.lastname: Orrego
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (ICP), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie, 28049, Madrid, Spain.
authors.forename: Sandro Martins
authors.identifier: http://orcid.org/0000-0002-4863-7137
authors.initials: SM
authors.lastname: Oliveira
authors.affiliation: Departamento de Biotecnología y Microbiología de Alimentos, Instituto de Investigación en Ciencias de los Alimentos (CIAL), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, 28049, Madrid, Spain.
authors.forename: Benevides Costa
authors.initials: BC
authors.lastname: Pessela
authors.affiliation: Departamento de Bioquímica e Microbiologia, Instituto de Biociências, Univ Estadual Paulista (UNESP), Caixa Postal 199, Av. 24 A, 1515, Bela Vista, Rio Claro, SP, 13506-900, Brazil.
authors.forename: Eleonora Cano
authors.initials: EC
authors.lastname: Carmona
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis (ICP), Consejo Superior de Investigaciones Científicas (CSIC), Campus Universidad Autónoma de Madrid (UAM), Cantoblanco, Calle de Marie Curie, 28049, Madrid, Spain.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Guisan
mesh_terms: D002413:Cations, Divalent; D019343:Citric Acid / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D006027:Glycosides / Q000737:chemistry; D006038:Glyoxylates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007854:Lead / Q000737:chemistry; D008628:Mercury / Q000737:chemistry; D008970:Molecular Weight; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology*; D012685:Sepharose / Q000737:chemistry; D013379:Substrate Specificity; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D002413:Cations, Divalent; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D006027:Glycosides; D006038:Glyoxylates; C070399:glyoxyl agarose; C008646:4-nitrophenyl beta-D-xyloside; D019343:Citric Acid; D007854:Lead; D003035:Cobalt; D003300:Copper; D012685:Sepharose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; D008628:Mercury
keywords: Enzyme characterization; Enzyme immobilization; Enzyme purification; Enzyme stabilization; Penicillium janczewskii; β-Xylosidases