Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii β-glucosidase

• Published in: Applied microbiology and biotechnology 2017-02, Vol.101 (4), p.1455-1463
• Main Authors: Sinha, Sushant K., Goswami, Shubhasish, Das, Shibashis, Datta, Supratim
• Format: Article
• Language: English
• Subjects: Bacteria - enzymology; beta-Glucosidase - metabolism; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Enzyme Stability - physiology; Escherichia coli; Life Sciences; Microbial Genetics and Genomics; Microbiology; Protein Engineering; Trichoderma viride
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-016-7904-y

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 p NPGlc and a half-life of 1.13 h when assayed with p NPGlc 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 p NPGlc, 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.