Production, characterization, in silico and crystal structure analyses of recombinant endo-β-1,3-glucanase of family 81 glycoside hydrolase (GH81) from Clostridium thermocellum ATCC 27405 and synthesis of laminarioligosaccharides for prebiotic applications

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The thermophilic bacterium Clostridium thermocellum contains multienzyme complex called cellulosome which is known to degrade recalcitrant substrates. In this study one of the cellulosomal enzymes, β-1,3-glucanase (CtLam81A) of family 81 GH from Clostridium thermocellum was explored. CtLam81A was highly thermostable and displayed endo β-1,3-glucanase activity. The in silico and X-ray crystal structure of CtLam81A showed N-terminal β-sandwich domain, a (α/α)6 domain and a short β-sandwich domain at C-terminal. Structural analysis of CtLam81A displayed the inverting hydrolytic mechanism. The docking of laminari-oligosaccharides to CtLam81A revealed that the active site can occupy maximum 5 glucose residues of β-1,3-glucan. The aromatic amino acid residues create the binding pocket at active site of CtLam81A to hold the ligand. The laminari-oligosaccharides generated by hydrolysis of curdlan by CtLam81A displayed prebiotic properties. They showed resistance or low digestibility against artificial gastric juice, intestinal fluid and α-amylase than inulin indicating their bioavailability to the probiotic bacteria present in the gastrointestinal tract of human. Therefore, laminari-oligosaccharides can be used for functional food additives.
Supervisor: Arun Goyal