A Novel Three Domains Glycoside Hydrolase Family 3 from Sclerotinia sclerotiorum Exhibits beta-Glucosidase and Exoglucanase Activities: Molecular, Biochemical, and Transglycosylation Potential Analysis

作者：Chahed Haifa^*; Ezzine Aymen; Ben Mlouka Mohamed Amine; Rihouey Christophe; Hardouin Julie; Jouenne Thierry; Marzouki M Nejib

来源：Molecular Biotechnology, 2015, 57(11-12): 993-1002.

DOI：10.1007/s12033-015-9892-z

• Glycoside hydrolase
• beta-glucosidase
• Transglycosylation
• Modeling
• Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes. We report here the purification and the biochemical characterization of a new beta-glucosidase from S. sclerotiorum which belongs to the family 3 of glycoside hydrolases and that was named as SsBgl3. After two size-exclusion chromatography steps, purified protein bands of 80 and 90 kDa from SDS-PAGE were subjected to a mass spectrometry analysis. The results displayed four peptides from the upper band belonging to a polypeptide of 777 amino acids having a calculated molecular weight of 83.7 kDa. Biochemical analysis has been carried out to determine some properties. We showed that this SsBgl3 protein displayed both beta-glucosidase and exoglucanase activities with optimal activity at 55 A degrees C and at pH 5. The transglycosylation activity was investigated using gluco-oligosaccharides TLC analysis. The molecular modeling and comparison with different crystal structures of beta-glucosidases showed that SsBgl3 putative protein present three domains. They correspond to an (alpha/beta)(8) domain TIM barrel, a five-stranded alpha/beta sandwich domain (both of which are important for active-site organization), and a C-terminal fibronectin type III domain. Enzyme engineering will be soon investigated to identify the key residues for the catalytic reactions.

• 出版日期2015-12