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Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability

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Abstract

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.

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Abbreviations

AaCel9A:

Alicyclobacillus acidocaldarius endoglucanase Cel9A

AaCel9AΔN:

AaCel9A without Ig-like domain

CBD:

carbohydrate binding domain

CMC:

Carboxymethylcellulose

DNS:

3,5-Dinitrosalicylic acid

GH:

Glycoside hydrolase

Ig-like:

Immunoglobulin- like

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Acknowledgments

We thank Professor S. Moréra (from Laboratoired’Enzymologie et BiochimieStructurales (LEBS), CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France) for the gift of pDEST17-AaCel9A. The authors express their gratitude to the research council of Azarbaijan Shahid Madani University for the financial support during the course of this project.

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Authors and Affiliations

  1. Department of Cellular and Molecular Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran

    Fereshteh S. Younesi, Mohammad Pazhang, Saeed Najavand, Parastou Rahimizadeh & Mohsen Akbarian

  2. Department of Nanobiotechnology, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran

    Mehdi Mohammadian

  3. Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran, Iran

    Khosro Khajeh

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  1. Fereshteh S. Younesi
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Correspondence to Mohammad Pazhang.

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Younesi, F.S., Pazhang, M., Najavand, S. et al. Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability. Mol Biotechnol 58, 12–21 (2016). https://doi.org/10.1007/s12033-015-9900-3

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