Studies towards Understanding the Molecular Basis of glucose Tolerance in a GH1 β-glucosidase from Halothermothrix orenii

Das, Shibashis (2016) Studies towards Understanding the Molecular Basis of glucose Tolerance in a GH1 β-glucosidase from Halothermothrix orenii. Masters thesis, Indian Institute of Science Education and Research Kolkata.

PDF (MS dissertation of Shibashis Das (11MS076)) Shibashis_Das_11MS076_Master_thesis.pdf - Submitted Version Restricted to Repository staff only Download (4MB)

Abstract

β-glucosidase (EC 3.2.1.21) cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. Glucose inhibition is a bottleneck in the enzymatic degradation of cellulose. B8CYA8 is a GH1 β-glucosidase from Halothermothrix orenii that retain 100% activity 1.5 M of glucose. Such high tolerance is needed to process cellulose for high gravity fermentation where high alcohol yield is achieved using more than 1M concentration of glucose. To find the reason behind glucose tolerance in the β-glucosidases several residues of these proteins were mutated. In two such mutant B8CYA8_W122F where the hydrophobicity of the residue was increased, no changes in Km were observed in the range of 0-500 mM glucose and the mutants could retain 100% activity up to 2.5 M glucose compared to 1.2 M of wild-type. When hydrophobic W168 was muted to hydrophilic arginine in B8CYA8_W168R, a drastic increase in Km was observed with respect to increasing glucose concentration. The Km at 500 mM was almost 10 times more than at 0 mM. The change in Km followed the pattern shown by wild-type upon varying the size of the mutated residue without a change in the hydrophobic nature of the residue. The insights obtained was then used to engineer a glucose tolerant BG from Agrobacterium tumifaciens BG, H0HC94. H0HC94_C174V retains 90% of its specific activity at 1 M glucose compared to the wild-type enzyme which loses 60% of its specific activity in 1 M of glucose.

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