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Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance

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Abstract

Alkaline pectin lyase (PNL) shows potential as a biological control agent against several plant diseases. We isolated and characterized a new Bacillus clausii strain that can produce 4,180 U/g of PNL using sugar beet pulp as a carbon source and inducer. The PNL was purified to apparent homogeneity using ultrafiltration, ammonium sulfate fractionation, DEAE Sepharose Fast Flow, and Sephadex G-75 gel filtration. The purified PNL was found to be a monomeric protein with a molecular weight of 35 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It demonstrated optimal activity with K m of 0.87 mg/ml at pH 10.0 and 60 °C. The enzyme is stable in the pH range of 8.0–10.0 and temperature ≤40 °C. Ca2+ was found to stimulate the enzymatic activity of the PNL by up to 410 %. Mass spectrometric results gave 38 % match coverage with pectate lyase from B. clausii KSM-K16 (gi|56961845). The PNL was found to elicit disease resistance in cucumber seedlings, suggesting that it may have applications in biocontrol and sustainable agriculture.

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Acknowledgments

This work was supported by Subsidy for Outstanding People of Beijing (No. 2011A005022 000004), the “Knowledge Innovation” Program of Chinese Academy of Sciences (No. KZZD-EW-09-3), the National Key Technology R&D Program of China (No. 2009BAC57B02), and Beijing Municipal Commission of Education (No. KM200811417006).

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Authors and Affiliations

  1. College of Arts and Sciences of Beijing Union University, Beijing, 100191, China

    Zuming Li

  2. Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

    Zuming Li, Zhihui Bai, Baoguo Zhang & Hongxun Zhang

  3. Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Baojv Li

  4. School of Chemical Engineering, the University of Adelaide, Adelaide, SA 5005, Australia

    Bo Jin

  5. Department of Physics and Astronomy, University of Manitoba, Winnipeg, R3T 2N2, Canada

    Michael Zhang & Francis Lin

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  1. Zuming Li
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  2. Zhihui Bai
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  4. Baojv Li
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Correspondence to Zhihui Bai.

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Li, Z., Bai, Z., Zhang, B. et al. Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance. Appl Biochem Biotechnol 167, 2241–2256 (2012). https://doi.org/10.1007/s12010-012-9758-9

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  • DOI: https://doi.org/10.1007/s12010-012-9758-9

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