Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.This work was funded by grant BIO2013-48779-C4-3-R from Spain's "Secretaría de Estado de Investigación, Desarrollo e Innovación". NR was supported by a Marie Curie ITN fellowship from the European Union within the Project Leangreenfood. DTP was supported by a FPU fellowship from "Ministerio de Economía y Competitividad".USD 1,495 APC fee funded by the EC FP7 Post-Grant Open Access PilotPeer reviewe

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