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Biochemical Characterization, Molecular Cloning, and Structural Modeling of an Interesting β-1,4-Glucanase from Sclerotinia Sclerotiorum

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Abstract

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-β-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC–MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase.

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Acknowledgments

We thank Dr. Thierry JOUENNE, director of the Laboratory of Polymers, Biopolymers and Surfaces in Rouen for providing the access to make mass spectrometry analysis and helpful discussions. This work is entirely financed by the Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB) in the National Institute of Applied Sciences and Technology of Tunis, University of Carthage. The Tunisian Ministry of High Education, Scientific Research and Technology is gratefully acknowledged.

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  1. Aymen Ezzine

    Present address: Laboratoire de Chimie Physique, Université de Paris-Sud, Bâtiment 350 - Campus d′Orsay. 15, Avenue Jean PERRIN, 91405, Orsay Cedex, France

Authors and Affiliations

  1. Laboratoire d’Ingénierie des Protéines et des Molécules Bioactives (LIP-MB), National Institute of Applied Sciences and Technology, University of Carthage, 1080, Tunis Cedex, Tunisia

    Haifa Chahed, Aymen Ezzine & Mohamed Najib Marzouki

  2. Laboratoire Polymères Biopolymères Surfaces (PBS), UMR 6270 CNRS, Université de Rouen, 76821, Mont-Saint-Aignan Cedex, France

    Amine Ben Mlouka, Julie Hardouin & Thierry Jouenne

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  1. Haifa Chahed
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Correspondence to Aymen Ezzine.

Electronic supplementary material

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12033_2013_9714_MOESM1_ESM.docx

Collected data for mass spectrometry analysis. Raw data files were processed using Proteome Discoverer 1.3 software (Thermo Scientific). Peak lists were searched using the MASCOT search engine (Matrix Science). (DOCX 1,426 kb)

12033_2013_9714_MOESM2_ESM.docx

cDNA and genomic DNA sequences of the Endo2 β-1,4-endoglucanase of S. sclerotiorum. Introns are shown with silver background. Arrows indicates primers localization. (DOCX 15 kb)

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Chahed, H., Ezzine, A., Mlouka, A.B. et al. Biochemical Characterization, Molecular Cloning, and Structural Modeling of an Interesting β-1,4-Glucanase from Sclerotinia Sclerotiorum . Mol Biotechnol 56, 340–350 (2014). https://doi.org/10.1007/s12033-013-9714-0

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