Thermal stability of the mutants obtained by rational design.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 45 (dark grey bars) or 80 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p

Structural detail in the vicinity of residues critical for GOX stability.

<p>Relevant interactions are depicted with dashed lines. (A) T554 in wild-type enzyme (left panel) and M554 in T554M mutant (right panel). Θ = 60°; d = 5 Å. (B) Q345 in wild-type enzyme. (C) R90 and E509 in the double mutant Q90R/Y509E. The subunit of origin is indicated in parenthesis. N-acetyl-glucosamine modification is colored in orange.</p

Mutations designed to introduce new salt bridges in <i>A</i>. <i>niger</i> GOX.

<p>(A) Q469K/L500D; (B) Q142R/L569E; (C) Q90R/Y509E; (D) H172K/H220D; (E) H447K; (F) Q345K. In A-D, sequence alignments with homologous enzymes from thermo-tolerant organisms are shown. Sequence codes are as follows: An_GOX: GOX from <i>A</i>. <i>niger</i> (Uniprot code P13006); Af_GOX: GOX from <i>A</i>. <i>fumigatus</i> (Uniprot code BOXU64); Hrt_GMC: glucose-methanol-choline oxidoreductase from <i>Halorubrum tebenquichense</i> (Genbank code WP_006628503.1); Htt_GMC: glucose-methanol-choline oxidoreductase from <i>Haloterrigena thermotolerans</i> (Genbank code WP_006648055.1); Tc_GMC: Glucose-methanol-choline oxidoreductase from <i>Thermomonospora curvata</i> (Uniprot code D1A2Y2); Tb_GMC: Glucose-methanol-choline oxidoreductase from <i>Thermobispora bispora</i> (Uniprot code D6Y5M6). Residues involved in the predicted salt bridges in An_GOX-homologous enzymes are highlighted in blue (cationic partner) and red (anionic partner). Panels on the right show details of An_GOX structure (PDB code 1CF3) and homology-based models of Af_GOX (green) and Htt_GOX (orange). An_GOX residues to be mutated and those involved in putative salt bridges in the homologues are shown. Panels E and F display the position of two single mutations. The residue to be mutated and the putative partner to form a salt bridge are shown. The two subunits of An_GOX structure are depicted in grey and blue.</p

Analysis of glycosylation pattern and specific activity of selected GOX mutants.

<p>(A) Proteins released to the culture medium were analyzed without (C) or with (E) EndoH treatment. GOX was identified as a differential band compared to a yeast control transformed with the same plasmid lacking the GOX gene. Migration of the deglycosylated GOX (dGOX) in the E lanes is indicated by an arrow and that of the glycosylated GOX (gGOX) in the C lanes is shown by a bracket. (B) Relative intrinsic activity of GOX mutants. Error bars represent standard deviation of analytical triplicates. Significant differences (p < 0.01) with the wild-type enzyme are indicated by asterisks.</p

Thermal stability of the enzymes with combined mutations.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 25 (dark grey bars) or 45 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p

Thermal stability of T554M mutant obtained by random mutagenesis.

<p>(A) Relative initial GOX activity in the culture medium of the yeast transformants. (B) Residual GOX activity after incubation at 60°C for 45 (dark grey bars) or 80 (light grey bars) minutes. Error bars represent standard deviation of triplicates. Significant differences (p < 0.05 or p < 0.01) with the wild-type enzyme are indicated by one or two asterisks, respectively.</p