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Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii

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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+2 and 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.

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Abbreviations

p-NPX:

p-Nitrophenyl β-D-xylopyranoside

PEG:

Polyethylene glycol

PEI:

Polyethylenimine

SDS:

Sodium dodecyl sulfate

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Acknowledgements

This work was partially sponsored by FAPESP (Project 2010/16582-0) and the Spanish Ministry of Science and Innovation (Project BIO-2012-36861). C.R.F.T. gratefully acknowledges PROPe/UNESP and CAPES/Ministry of Education, Brazil, through the Program Science Without Borders for the postdoctoral scholarship (3134-13-0).

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Authors and Affiliations

  1. 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

    César Rafael Fanchini Terrasan, Maria Romero-Fernández, Alejandro H. Orrego, Sandro Martins Oliveira & José Manuel Guisan

  2. 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

    César Rafael Fanchini Terrasan & Eleonora Cano Carmona

  3. 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

    Benevides Costa Pessela

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  1. César Rafael Fanchini Terrasan
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  2. Maria Romero-Fernández
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Correspondence to César Rafael Fanchini Terrasan.

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ESM 1

Stability of BXYL I from P. janczewskii in pH 10.0 medium with polyethylene glycol. Incubation was carried out with 0.1 M sodium bicarbonate buffer pH 10.0 containing 50% (w/v) PEG MW 6000 and 35,000 in an ice-bath and at room temperature. The activity was assayed in 0.05 M sodium acetate buffer pH 5.0 at 25 °C. Incubation was carried out at room temperature (dashed lines) and in an ice-cold bath (full lines). β-xylosidase activity (%) in medium with PEG (▲, ▵) 6000 and (■, □) PEG 35,000 (JPEG 107 kb)

High Resolution Image (EPS 6 kb)

ESM 2

Immobilization course of BXYL I from P. janczewskii on glyoxyl agarose. Immobilization was performed in 0.1 M sodium bicarbonate buffer pH 10.0 containing 50% (w/v) PEG MW 35,000. Immobilization was initially performed in an ice-cold bath and subsequently the temperature was allowed to gradually increase to room temperature (≈ 25 °C). The activity was assayed in 0.05 M sodium acetate buffer pH 5.0 at 25 °C. β-xylosidase activity (%) in the (●) control, (■) supernatant and (▲) suspension. Vertical dashed line indicates the change in incubation temperature (JPEG 120 kb)

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Terrasan, C.R.F., Romero-Fernández, M., Orrego, A.H. et al. Immobilization and Stabilization of Beta-Xylosidases from Penicillium janczewskii . Appl Biochem Biotechnol 182, 349–366 (2017). https://doi.org/10.1007/s12010-016-2331-1

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