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Characterization of a Neutral and Thermostable Glucoamylase from the Thermophilic Mold Thermomucor indicae-seudaticae: Activity, Stability, and Structural Correlation

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Abstract

Glucoamylase from the thermophilic mold Thermomucor indicae-seudaticae was purified by anion exchange and gel filtration chromatographic techniques using a fast protein liquid chromatographic system. The structure and thermal stability of this unique ‘thermostable and neutral glucoamylase’ were analyzed by circular dichroism (CD). T. indicae-seudaticae glucoamylase (TGA) contained typical aromatic amino acid (tryptophan/tyrosine) fingerprints in its tertiary structure. Analysis of the far-UV CD spectrum at pH 7.0 and 25 °C revealed the presence of 45% α-helix, 43% β-sheet, and 12% remaining structures. The α-helix content was highest at pH 7.0, where glucoamylase is optimally active. This observation points towards the possible (α/α)6 barrel catalytic domain in TGA, as reported in microbial glucoamylases. Thermal denaturation curves of the pure protein at different pH values revealed maximum stability at pH 7.0, where no change in the secondary structure was observed upon heating in the temperature range between 20 °C and 60 °C. The observed midpoint of thermal denaturation (T m) of glucoamylase at pH 7.0 was 67.1 °C, which decreased on either sides of this pH. Thermostability of TGA enhanced in the presence of starch (0.1%) as no transition curve was obtained in the temperature range between 20 °C and 85 °C. The only product of TGA action on starch was glucose, and it did not exhibit transglycosylation activity even at 40% glucose that can also be considered as an advantage during starch saccharification.

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Abbreviations

TGA:

Thermomucor indicae-seudaticae glucoamylase

T m :

midpoint of thermal denaturation

CD:

circular dichroism

\( f_{\alpha } \), \( f_{\beta } \), ft and fr:

fractions of α-helix, β-sheet, β-turn, and random coil, respectively

f D :

fraction of denatured protein

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Acknowledgments

PK is grateful to the Council of Scientific and Industrial Research, Government of India, for Junior/Senior Research Fellowship during the course of this investigation. This work was partially supported by the research grants from the Council of Scientific & Industrial Research and Department of Science and Technology, Government of India.

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Authors and Affiliations

  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110 021, India

    Pardeep Kumar & T. Satyanarayana

  2. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110 025, India

    Asimul Islam & Faizan Ahmad

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  1. Pardeep Kumar
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  2. Asimul Islam
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  3. Faizan Ahmad
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  4. T. Satyanarayana
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Correspondence to T. Satyanarayana.

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Kumar, P., Islam, A., Ahmad, F. et al. Characterization of a Neutral and Thermostable Glucoamylase from the Thermophilic Mold Thermomucor indicae-seudaticae: Activity, Stability, and Structural Correlation. Appl Biochem Biotechnol 160, 879–890 (2010). https://doi.org/10.1007/s12010-009-8666-0

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