title: Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii β-glucosidase.
abstract: β-glucosidase (EC 3.2.1.21; BG) cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345 μmol/min/mg on pNPGlc and a half-life of 1.13 h when assayed with pNPGlc at pH 7.1 and 70 °C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.
authors.affiliation: Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India.
authors.forename: Sushant K
authors.initials: SK
authors.lastname: Sinha
authors.affiliation: Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India.
authors.forename: Shubhasish
authors.initials: S
authors.lastname: Goswami
authors.affiliation: Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India.
authors.forename: Shibashis
authors.initials: S
authors.lastname: Das
authors.affiliation: Protein Engineering Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India. supratim@iiserkol.ac.in.|Centre for Advanced Functional Materials, Indian Institute of Science Education and Research Kolkata, Mohanpur, 741246, India. supratim@iiserkol.ac.in.
authors.forename: Supratim
authors.initials: S
authors.lastname: Datta
mesh_terms: D001419:Bacteria / Q000201:enzymology*; D004795:Enzyme Stability / Q000502:physiology; D015202:Protein Engineering; D001617:beta-Glucosidase / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase
keywords: Aglycone; Half-life; Halophile; Protein engineering; Thermostability; β-glucosidase