2-Keto-D-Gluconate-Yielding Membrane-Bound D-Glucose Dehydrogenase from Arthrobacter globiformis C224: Purification and Characterization
Abstract
:1. Introduction
2. Results and Discussion
2.1. Extraction and Purification of GlcDH from A. globiformis C224
Step | Protein (mg) | Enzyme Activity (Units) | Specific Activity (Units/mg) | Recovery (%) | Purification (Fold) |
---|---|---|---|---|---|
Membrane fraction | 9758.1 ± 121.3 | 4730.6 ± 96.5 | 0.5 | 100.0 | 1.0 |
Triton X-114 phase separation | 6004.5 ± 96.1 | 4396.4 ± 79.8 | 0.7 | 92.9 | 1.5 |
Acetone precipitation | 1103.7 ± 30.6 | 1524.9 ± 34.2 | 1.4 | 32.2 | 2.9 |
PEG precipitation | 408.8 ± 17.4 | 846.3 ± 12.3 | 2.1 | 17.9 | 4.3 |
Ethanol precipitation | 33.6 ± 1.2 | 268.2 ± 6.4 | 7.9 | 5. 7 | 16.6 |
Hydroxylapatite fraction | 1.8 ± 0.1 | 159.4 ± 2.1 | 88.1 | 3.4 | 183.5 |
2.2. Type Identification of GlcDH from A. globiformis C224
Protein | Similar Sequence in the Database of the NCBI Blast | Strains | Mass (Da) | Score |
---|---|---|---|---|
Gi|104780479 glucose dehydrogenase | R.LLALDPDTGAEIWR.Y | Pseudomonas entomophila L48 | 87,067 | 142 |
R.GIGPFTAGGYYSTSPAAITR.S | ||||
Gi|339489095 glucose dehydrogenase | R.TEHGDRYSPLR.Q | Pseudomonas putida S16 | 86,935 | 117 |
R.LLALDPDTGAEIWR.F | ||||
R.GVSYYDENRYVSR.D | ||||
Gi|167035354 PQQ-dependent glucose dehydrogenase | R.TEHGDRYSPLR.Q | Pseudomonas putida GB-1 | 86,939 | 114 |
R.LLALDPDTGAEIWR.F | ||||
R.GVSYYDENRYVSR.D | ||||
Gi|26988177 glucose dehydrogenase | R.LLALDPDTGAEIWR.Y | Pseudomonas putida KT2440 | 86,926 | 100 |
R.GVSYYDENRYVSR.D |
2.3. Enzymatic Properties of Purified Membrane-Bound GlcDH from A. globiformis C224
2.3.1. Substrate Specificity
Substrate | Concentration of Substrate (mM) | Membrane Enzyme (U/mL) | Purified EnzymeRelative Activity (%) |
---|---|---|---|
d-Glucose | 33.0 | 21.9 ± 0.9 | 100.0 ± 1.6 |
d-Gluconate | 33.0 | 82.2 ± 3.8 | 0 |
Maltose | 33.0 | 3.1 ± 0.1 | 17.0 ± 0.8 |
d-Sorbose | 33.0 | 0 | 0 |
d-Galactose | 33.0 | 3.8 ± 0.1 | 20.0 ± 0.9 |
d-Mannose | 33.0 | 0 | 0 |
d-Fructose | 33.0 | 0 | 0 |
d-Arabinose | 33.0 | 2.1 ± 0.1 | 8.0 ± 0.5 |
Malic acid | 33.0 | 0 | 0 |
Sucrose | 33.0 | 0 | 0 |
Citric acid | 33.0 | 0 | 0 |
d-Xylose | 33.0 | 5.3 ± 0.2 | 22.0 ± 0.9 |
2.3.2. Kinetic Studies of GlcDH from A. globiformis C224
Single Substrate Kinetics
Substrate | Km (mM) | Vmax (μmol/mg·min) |
---|---|---|
d-Glucose | 0.21 ± 0.01 | 192.31 ± 9.45 |
d-Xylose | 0.34 ± 0.02 | 44.05 ± 1.94 |
d-Galactose | 0.46 ± 0.02 | 18.32 ± 0.76 |
Maltose | 0.59 ± 0.03 | 19.05 ± 0.85 |
Dual Substrates Kinetics
Inhibition by Gluconic Acid
2.3.3. Effect of pH on GlcDH Activity and Stability
Properties | Escherichia coli [11] | Gluconobacter Suboxydans [12] | Bacillus Thuringiensis [13] | Pseudomonas sp. [14] | Acinetobacter Calcoaceticus [15] | Arthrobacter Globiformis(This Study) |
---|---|---|---|---|---|---|
Molecular mass of subunits (Da) | 88,000 | 87,000 | 25,000 26,000 | 90,000 | 47,500 48,000 | 87,000 |
Optimum pH with ferricyanide | 3.5 | 3.0 | ND | 4.5 | ND | 5.0 |
With DCIP-PMS | 6.0 | 6.0 | 8.0 | 6.0 | 6.0 | ND |
Optimum temperature (°C) | ND | ND | 55 | ND | ND | 45 |
Substrate range | Glucose; Mannose; Galactose; Fructose; Rhamnose; Xylose | Glucose; Maltose | Glucose; 2-d-deoxy-d-glucose | Glucose; Mannose; Galactose; Xylose; Maltose; Rhamnose | Glucose; Xylose; Arabinose; Lactose; Galactose; Melibiose; Cellobiose; Maltose | Glucose; Xylose; Galactose; Maltose |
Km (mM) | ND | ND | 14 (Glucose, pH 8.0); 12.2 (2-d-deoxy-d-glucose, pH 8.0) | 0.69 (Glucose, pH 4.5); 1.6 (Glucose, pH 6.0) | 22 (Glucose, pH 6.0) | 0.21 (Glucose, pH 5.0); 0.34 (Xylose, pH 5.0); 0.46 (Galactose, pH 5.0); 0.59 (Maltose, pH 5.0) |
2.3.4. Effect of Temperature on GlcDH Activity and Stability
2.3.5. Effect of Organic Solvents on GlcDH Activity
2.3.6. Effect of Metal Ions or EDTA on GlcDH Activity
3. Experimental Section
3.1. Chemicals
3.2. Microorganism and Medium
3.3. Preparation of Cell Culture
3.4. Extraction and Purification of GlcDH from A. globiformis C224
3.5. Polyacrylamide Gel Electrophoresis
3.6. MALDI-TOF-MS Analysis
3.7. Assay of GlcDH Activity and Protein Determination
3.8. Enzymatic Properties
3.8.1. Substrate Specificity
3.8.2. Effect of pH, Temperature, Organic Solvents or Metal Ions on GlcDH Activity
3.9. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Xue, Q.; Wei, Z.; Sun, W.; Cui, F.; Yu, S.; Zhou, Q.; Liu, J. 2-Keto-D-Gluconate-Yielding Membrane-Bound D-Glucose Dehydrogenase from Arthrobacter globiformis C224: Purification and Characterization. Molecules 2015, 20, 846-862. https://doi.org/10.3390/molecules20010846
Xue Q, Wei Z, Sun W, Cui F, Yu S, Zhou Q, Liu J. 2-Keto-D-Gluconate-Yielding Membrane-Bound D-Glucose Dehydrogenase from Arthrobacter globiformis C224: Purification and Characterization. Molecules. 2015; 20(1):846-862. https://doi.org/10.3390/molecules20010846
Chicago/Turabian StyleXue, Qing, Zhuan Wei, Wenjing Sun, Fengjie Cui, Silian Yu, Qiang Zhou, and Jingze Liu. 2015. "2-Keto-D-Gluconate-Yielding Membrane-Bound D-Glucose Dehydrogenase from Arthrobacter globiformis C224: Purification and Characterization" Molecules 20, no. 1: 846-862. https://doi.org/10.3390/molecules20010846