Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability

Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (KM) and nearly four-fold greater catalytic rate (kcat). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in kcat at the expense of a 3% loss of substrate affinity (increase in apparent KM for glucose) resulting in a specify constant (kcat/KM) of 23.8 (mM−1 · s−1) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM−1 · s−1, respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain

Structural comparison of the GOx FAD adduct (1cf3) with the FAD-peroxy adduct of choline oxidase (2jbv).

<p>Panel A has illustrations of GOx at two different angles. Panel B illustrates the choline oxidase FAD-peroxy adduct at two different angles. Thr 132 (GOx) and its homologous amino acid Ile 103 (choline oxidase) are labeled. The peroxy adduct is colored green. Other oxygens are red, carbons are teal, nitrogens are blue, and phosphorous atoms are tan.</p

Electrochemical GOx activity assay: H₂O₂ concentration calibration and assay controls.

<p>A) Steady state current response vs. H₂O₂ concentration. Pt working electrode held at +700 mV vs. Ag/AgCl, 6000 rpm, in 100 mM NaPB, pH 6.8. Error bars are the standard deviation of 3 independent measurements. Inset: Lower H₂O₂ concentration segment of plot. B) Current response upon a 25 µL injection of sample prepared from yeast cultures in which GOx expression was induced (red trace) or uninduced (blue trace), into 100 mM NaPB, pH 6.8 with 100 mM glucose. Injection of sample prepared from induced GOx expression yeast culture into 100 mM NaPB, pH 6.8, without glucose (green trace). Pt working electrode held at +700 mV vs. Ag/AgCl, 6000 rpm.</p

View of amino acid mutations that enhanced GOx kinetic activity in relation to the FAD cofactor: T132S, T56V, and V42T.

<p>The monomer protein is shown as ribbons. The FAD group and mutated amino acid residues are shown as space-filling models. The FAD binding peptide region, containing amino acids T56V and V42T, is colored red.</p

Western blot of Ni-NTA affinity purified yeast culture media.

<p>Bands observed via anti-V5 epitope-AP antibody labeling. Lane 1: Sample from culture with induced GOx expression; Lane 2: Sample from uninduced culture.</p

A cartoon view of GOx monomer, with the protein shown as ribbons and the FAD groups shown as space-filling models.

<p>Residues that were targeted for mutagenesis are also shown as space-filling models and labeled. The numbering used is from the <i>A. niger</i> protein sequence and includes the 22 amino acid leader peptide. Residue N536 is obscured by residues T132, R534, and T537.</p

Kinetic rate parameters of parental and mutant GOx stains for D (+) glucose oxidation as determined via initial rate electrochemical measurements.

1<p>± values are 95% confidence intervals from a non-linear least squares regression fit of initial rate data to the Michaelis-Menton equation.</p>2<p><i>k</i>_cat defined per mol native GOx.</p>3<p>Negative change in <i>K</i>_M denotes higher affinity for substrate.</p>4<p>Values taken from reference 14.</p

Thermal stability of parental and mutant GOx stains incubated at 50 °C.

1<p>Error corresponds to the standard deviation of GOx kinetic rate measurements performed in triplicate.</p>2<p>Correlation coefficients obtained from an exponential least squares regression fit of GOx kinetic rates measured in triplicate vs. time.</p

Comparison between Amplex Red and ABTS GOx activity assays.

<p>A) Amplex Red and B) ABTS assay absorbance vs. GOx concentration standard curves. Glucose concentration was 50 mM. Error bars are the standard deviation of 3 independent measurements. Comparison of activity assay results for mutant GOx stains using C) Amplex Red or D) ABTS assay. Each 96 well plate contained 96 different mutant GOx samples that were loaded in identical wells between plates.</p