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Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-β-(1,4)-xylanase of low molecular weight

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Abstract

Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea, comprises eight pathogenic races and two pathotypes. Races 0 and 5, representative of the least virulent yellowing pathotype and the most virulent wilt pathotype, respectively, produced extracellular xylanases when grown on minimal medium supplemented with either 1% commercial birchwood xylan or 0.3% chickpea cell walls. The pattern of extracellular proteins analysed by denaturing polyacrylamide gel electrophoresis in the two media presented some minor but distinctive differences between fungal races. By preparative isoelectrofocusing, the xylanase activity in cell wall-culture filtrates could be resolved into basic and neutral fractions with pI values around to 10 and 8, respectively, whereas the xylan-culture filtrates contained an additional acidic fraction of pI around 4. A common major xylanase was purified 7-fold to homogeneity by cation-exchange chromatography and chromatofocusing. The purified xylanase has a molecular weight of 21.6 kDa, optimum pH and temperature of 5.5 and 55 °C, respectively, pI in the range of 8.2 to 9.0, and Km and Vmax values of 2.24 mg ml−1 (birchwood xylan as substrate) and 1200 nkat  mg−1 protein (72 U mg−1 protein), respectively. The enzyme has an endo mode of action, hydrolysing xylan to xylobiose and higher short-chain xylooligosaccharides without forming free xylose.

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Abbreviations

SDS-PAGE:

denaturing polyacrylamide gel electrophoresis

IEF:

isoelectrofocusing

U:

International unit of enzyme activity (μmol min−1)

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Authors and Affiliations

  1. Departamento de Bioquímica y Biología Molecular, ETSIAM, Universidad de Córdoba, Apartado 3048, 14080, Córdoba, Spain

    Inmaculada Jorge, Olga de la Rosa & Manuel Tena

  2. Instituto de Agricultura Sostenible, Consejo Superior de Investigaciones Científicas, Apartado 4084, 14080, Córdoba, Spain

    Juan A. Navas-Cortés & Rafael M. Jiménez-Díaz

  3. Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Apartado 3048, 14080, Córdoba, Spain

    Rafael M. Jiménez-Díaz

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  1. Inmaculada Jorge
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  2. Olga de la Rosa
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  3. Juan A. Navas-Cortés
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  4. Rafael M. Jiménez-Díaz
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  5. Manuel Tena
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Correspondence to Manuel Tena.

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Jorge, I., Rosa, O.d.l., Navas-Cortés, J.A. et al. Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-β-(1,4)-xylanase of low molecular weight. Antonie Van Leeuwenhoek 88, 48–59 (2005). https://doi.org/10.1007/s10482-004-7584-y

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