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Characterization of a Thermostable Family 1 Glycosyl Hydrolase Enzyme from Putranjiva roxburghii Seeds

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Abstract

A 66-kDa thermostable family 1 Glycosyl Hydrolase (GH1) enzyme with β-glucosidase and β-galactosidase activities was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family. N-terminal and partial internal amino acid sequences showed significant resemblance to plant GH1 enzymes. Kinetic studies showed that enzyme hydrolyzed p-nitrophenyl β-d-glucopyranoside (pNP-Glc) with higher efficiency (K cat/K m = 2.27 × 104 M−1 s−1) as compared to p-nitrophenyl β-d-galactopyranoside (pNP-Gal; K cat/K m = 1.15 × 104 M−1 s−1). The optimum pH for β-galactosidase activity was 4.8 and 4.4 in citrate phosphate and acetate buffers respectively, while for β-glucosidase it was 4.6 in both buffers. The activation energy was found to be 10.6 kcal/mol in the temperature range 30–65 °C. The enzyme showed maximum activity at 65 °C with half life of ~40 min and first-order rate constant of 0.0172 min−1. Far-UV CD spectra of enzyme exhibited α, β pattern at room temperature at pH 8.0. This thermostable enzyme with dual specificity and higher catalytic efficiency can be utilized for different commercial applications.

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Acknowledgments

CD and FPLC studies were performed at NMR facility and Macromolecular Crystallographic Unit respectively at Institute Instrumentation Centre (IIC), IIT Roorkee. Girijesh Kumar Patel and Bibekananda Kar gratefully acknowledge the financial support from Ministry of Human Resource Development and CSIR, Government of India, respectively.

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Authors and Affiliations

  1. Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee-247 667, Uttarakhand, India

    Girijesh Kumar Patel, Bibekananda Kar & Ashwani Kumar Sharma

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  1. Girijesh Kumar Patel
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  2. Bibekananda Kar
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  3. Ashwani Kumar Sharma
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Correspondence to Ashwani Kumar Sharma.

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Patel, G.K., Kar, B. & Sharma, A.K. Characterization of a Thermostable Family 1 Glycosyl Hydrolase Enzyme from Putranjiva roxburghii Seeds. Appl Biochem Biotechnol 166, 523–535 (2012). https://doi.org/10.1007/s12010-011-9445-2

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