Isolation and biochemical characterization of a glucose dehydrogenase from a hay infusion metagenome

Abstract

Glucose hydrolyzing enzymes are essential to determine blood glucose level. A high-throughput screening approach was established to identify NAD(P)-dependent glucose dehydrogenases for the application in test stripes and the respective blood glucose meters. In the current report a glucose hydrolyzing enzyme, derived from a metagenomic library by expressing recombinant DNA fragments isolated from hay infusion, was characterized. The recombinant clone showing activity on glucose as substrate exhibited an open reading frame of 987 bp encoding for a peptide of 328 amino acids. The isolated enzyme showed typical sequence motifs of short-chain-dehydrogenases using NAD(P) as a co-factor and had a sequence similarity between 33 and 35% to characterized glucose dehydrogenases from different Bacillus species. The identified glucose dehydrogenase gene was expressed in E. coli, purified and subsequently characterized. The enzyme, belonging to the superfamily of short-chain dehydrogenases, shows a broad substrate range with a high affinity to glucose, xylose and glucose-6-phosphate. Due to its ability to be strongly associated with its cofactor NAD(P), the enzyme is able to directly transfer electrons from glucose oxidation to external electron acceptors by regenerating the cofactor while being still associated to the protein.

Figure 1. Amino acid sequence alignment of the GDH1E5 with several glucose dehydrogenases and SDR enzymes.

The secondary structure of the GDH1E5 (CCK35875) was predicted by the online tool PSIPRED 3.0 and is indicated as arrows for β-strands and as cylinders for α-helices. Conserved structural residues of SDR enzymes are highlighted. Active site residues are colored in green, residues for co-factor binding in blue and conserved structural residues in yellow. The sequence of the GDH1E5 was aligned with Glucose 1-Dehydrogenases (gdhA, gdh, GlcDH) from Bacillus megaterium (P10528), Bacillus subtilis (P12310), Burkholderia xenovorans (YP_554854), Cytophaga hutchinsonii (YP_678271), and two additional short-chain dehydrogenases (SDR) from Pseudomonas syringae (YP_235378) and Exiguobacterium sibiricum (YP_001815303).

Figure 2. SDS-polyacrylamide gel electrophoresis (12%) of the purification of GDH1E5.

The purification of GDH1E5 was examined after each purification step by SDS-PAGE. Lane 1: Protein standards. Lane 2: Crude extract. Lane 3: Hydrophobic interaction chromatography. Lane 4: Gel filtration. Lane 5: Immobilized metal ion affinity chromatography.

Figure 3. Molecular weight detection of native GDH1E5.

A: Native polyacrylamide gelelectrophoresis of the size exclusion chromatography step in combination with a glucose dehydrogenase activity staining. Lane 1: marker proteins: Thyroglobulin (669 kDa), Catalase (232 kDa), Lactate-Dehydrogenase (140 kDa) and Albumin (66 kDa). Lane 2: GDH1E5 after size exclusion chromatography step stained with Coomassie. Lane 3: Activity staining of the native PAGE using glucose as substrate and DCPIP as colorimetric indicator and electron acceptor. Glucose-Dehydrogenase activity is indicated by a clear halo in regards to the decolorization of reduced DCPIP. B: Plot of the retention value Rf of the marker proteins and GDH1E5 versus the logarithm of their regarding molecular weight. C: Chromatogram of the purification step of GDH1E5 via size exclusion chromatography using HiLoad 16/60 Superdex 200 prep grade. D: Plot of partition coefficient K_AV from the derived elution volumes of the marker proteins (Ferritin 440 kDa, Aldolase 158 kDa, Conalbumin 75 kDa, Ovalbumin 44 kDa) and GDH1E5 versus the logarithm of their regarding molecular weight.

Figure 4. Absorbance spectra of GDH1E5 in absence and presence of glucose.

A: Absorbance spectrum of purified GDH1E5 in the absence of substrate and external electron acceptors compared to the absorbance spectrum of the oxidized co-factor molecule NADP⁺. B: Absorbance spectrum of purified GDH1E5 in the presence of glucose and absence of external electron acceptors compared to the absorbance spectrum of the reduced co-factor molecule NADPH.

Figure 5. The effect of temperature on the activity of GDH1E5.

The enzyme activity was assayed at various temperatures in the range of 5 to 60°C. All solutions were preincubated at the corresponding temperature and the enzymatic activity was assayed for 5 min at the respective temperature using DCPIP as electron acceptor. All data represent the average of triplicate determinations ± standard derivation.

Figure 6. Influence of different temperatures on the stability of GDH1E5.

The residual activity of the enzyme was assayed after incubation at various temperatures in the range of 30 to 50°C. The residual activity was determined for 5 min at 35°C using D-glucose as substrate and DCPIP as electron acceptor. All data represent the average of triplicate determinations ± standard derivation. A: Thermal stability of the GDH1E5 over a span of time from 0–55 h. B: Zoomed view of the stability of the GDH1E5 at temperatures from 40 to 50°C over a span of 9 h (boxed area in A).

Figure 7. pH profile of GDH 1E5.

The enzyme activity was assayed at various pH values in Britton & Robinson universal buffer in the range of pH 2–11 using DCPIP as electron acceptor. The enzymatic activity was assayed for 5 min at 35°C and the according pH using DCPIP as electron acceptor. All data represent the average of triplicate determinations ± standard derivation.

Figure 8. Effect of various metal ions on GDH1E5.

The influence of di- and trivalent cations on enzyme activity was investigated using D-glucose as substrate and DCPIP as electron acceptor. Residual activity was determined after enzyme preincubation for 30 min in the presence of 1 (black bars) and 5 mM (grey bars) of the corresponding ion in citrate buffer (pH 6.0). Afterwards enzymatic activity was determined in the presence of the cations using DCPIP at 35°C.

Figure 9. Effect of various detergents and organic solvents on GDH1E5.

The influence of detergents (A) and organic solvents with respective concentrations of 1% (w/v, v/v) and 10% (v/v) was investigated for enzyme activity using D-glucose as substrate and DCPIP as electron acceptor. Residual activity was determined after preincubation of the enzyme for 30 min in the presence of the corresponding detergents and organic solvents in citrate buffer (pH 6.0) and sodium phosphate buffer (pH 6.0) respectively. Afterwards enzymatic activity was determined in the presence of the detergents and organic solvents using DCPIP at 35°C.