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.

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

“…The synthetic gene corresponding to the β-glucosidase from H. orenii was constructed (BankIt1930137BG_Halotherm KU867899) as reported previously and expressed in Escherichia coli Top 10F’ cells (Life Technologies, La Jolla, CA) [14]. The cells were centrifuged at 4000 × g for 10 min at 4 °C and the pellet stored at −20 °C until purification of the protein.…”

“…B8CYA8 and mutants were purified using a protocol as detailed earlier, and purity was confirmed by 10 % SDS–PAGE [14]. The protein concentrations were determined by measuring the absorbance at 280 nm and using the extinction coefficient for respective enzyme variants calculated using the modified Edelhoch and Gill/Von Hippel methods on Expasy (http://web.expasy.org/protparam/).…”

“…The kinetic parameters of all the mutants were determined at various concentrations, ranging between 0.5 mM to 100 mM, of substrates p NPGlc and Clb as previously reported [14]. GraphPad PRISM version 7.0 (GraphPad Software, La Jolla, CA) was used to calculate all kinetic constants by a non-linear regression fit of the Michaelis-Menten equation.…”

“…These observations do not, however, explain the ability of the enzyme pocket to distinguish between the reaction product glucose and the substrate cellobiose. Our sequence comparisons with other highly glucose tolerant β-glucosidase such as O08324, A0A0F7KKB7, and Q8T0W7 suggest that many of the residues previously implicated for glucose tolerance are non-conserved and therefore those specific residues may not play a role in glucose tolerance [14-16].…”

“…In this study, we used a β-glucosidase (B8CYA8) from the thermophilic and halophilic bacteria Halothermothrix orenii [17]. It was previously reported that B8CYA8 efficiently converts lactose to different transglycosylated products and hydrolyzes cellobiose to glucose [14, 18, 19]. Here we report that B8CYA8 is tolerant to high concentrations of glucose.…”