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Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1.

Anjum Zia M - J Clin Biochem Nutr (2007)

Bottom Line: Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40 degrees C.The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol(-1) and free energy of denaturation 103.63 kJ mol(-1).These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry (Biochemistry), University of Agriculture, Faisalabad, Pakistan.

ABSTRACT
An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40 degrees C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol(-1) and free energy of denaturation 103.63 kJ mol(-1). These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays.

No MeSH data available.


Related in: MedlinePlus

Arrhenius plot for determination of activation energy for the oxidation of D-glucose.
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Figure 3: Arrhenius plot for determination of activation energy for the oxidation of D-glucose.

Mentions: Glucose oxidase from Aspergillus niger UAF-1 was active within the pH range of 4–8, while maximum activity was observed at pH 5.5 (Fig. 2). The activation energy (Ea) and optimum temperature of glucose oxidase was found to be 15.46 kJ mol−1 and 40°C respectively. It is obvious from the Arrhenius plot (Fig. 3) that the enzyme had a single conformation up to transition temperature. The change in pH affects the ionization of essential active site amino acid residues, which are involved in substrate binding and catalysis. The ionization of these residues may cause distortion of active site cleft and hence indirectly affect the enzyme activity. Our results favorably compare to those of Weibel and Bright [31], who reported that glucose oxidase is working in the pH range of 4–7 indicating 5.5 as optimum. Aspergillus niger UAF-1 enzyme showed a low Ea at 40°C that makes the enzyme superior to the enzyme from various other sources. Purified glucose oxidase having a protein content of 0.05 mg mL−1 was used for the kinetic and thermodynamic characterization. The Km and Vmax values obtained from Lineweaver-Burk plot were 2.56 mM and 43.5 U mg−1 protein (Fig. 4). Referring to properties of biotechnological relevance, the glucose oxidase of Aspergillus niger exhibited a high affinity for D-glucose as it has low Km value for the substrate as compared to a high Km value (0.033 M) of glucose oxidase isolated from the Aspergillus niger reported earlier [32]. This high substrate affinity and specificity, in addition to its long-term stability in the pH range 4–8, proved glucose oxidase of Aspergillus niger UAF-1 as a suitable biocatalyst for industrial applications. The rates of reaction of glucose oxidase from Aspergillus niger with glucose, 2-deoxyglucose, mannose, galactose and xylose have been measured and found that glucose reacts much faster than the other sugars with Km 0.11 M and Vmax of 2000 s−1M [33].


Thermal Characterization of Purified Glucose Oxidase from A Newly Isolated Aspergillus Niger UAF-1.

Anjum Zia M - J Clin Biochem Nutr (2007)

Arrhenius plot for determination of activation energy for the oxidation of D-glucose.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170954&req=5

Figure 3: Arrhenius plot for determination of activation energy for the oxidation of D-glucose.
Mentions: Glucose oxidase from Aspergillus niger UAF-1 was active within the pH range of 4–8, while maximum activity was observed at pH 5.5 (Fig. 2). The activation energy (Ea) and optimum temperature of glucose oxidase was found to be 15.46 kJ mol−1 and 40°C respectively. It is obvious from the Arrhenius plot (Fig. 3) that the enzyme had a single conformation up to transition temperature. The change in pH affects the ionization of essential active site amino acid residues, which are involved in substrate binding and catalysis. The ionization of these residues may cause distortion of active site cleft and hence indirectly affect the enzyme activity. Our results favorably compare to those of Weibel and Bright [31], who reported that glucose oxidase is working in the pH range of 4–7 indicating 5.5 as optimum. Aspergillus niger UAF-1 enzyme showed a low Ea at 40°C that makes the enzyme superior to the enzyme from various other sources. Purified glucose oxidase having a protein content of 0.05 mg mL−1 was used for the kinetic and thermodynamic characterization. The Km and Vmax values obtained from Lineweaver-Burk plot were 2.56 mM and 43.5 U mg−1 protein (Fig. 4). Referring to properties of biotechnological relevance, the glucose oxidase of Aspergillus niger exhibited a high affinity for D-glucose as it has low Km value for the substrate as compared to a high Km value (0.033 M) of glucose oxidase isolated from the Aspergillus niger reported earlier [32]. This high substrate affinity and specificity, in addition to its long-term stability in the pH range 4–8, proved glucose oxidase of Aspergillus niger UAF-1 as a suitable biocatalyst for industrial applications. The rates of reaction of glucose oxidase from Aspergillus niger with glucose, 2-deoxyglucose, mannose, galactose and xylose have been measured and found that glucose reacts much faster than the other sugars with Km 0.11 M and Vmax of 2000 s−1M [33].

Bottom Line: Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40 degrees C.The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol(-1) and free energy of denaturation 103.63 kJ mol(-1).These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry (Biochemistry), University of Agriculture, Faisalabad, Pakistan.

ABSTRACT
An intracellular glucose oxidase was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger UAF-1. The enzyme was purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion exchange and gel filtration chromatography. The enzyme showed high affinity for D-glucose with a Km value of 2.56 mM. The enzyme exhibited optimum catalytic activity at pH 5.5. Temperature optimum for glucose oxidase, catalyzed D-glucose oxidation was 40 degrees C. The enzyme showed a high thermostability having a half-life 30 min, enthalpy of denaturation 99.66 kJ mol(-1) and free energy of denaturation 103.63 kJ mol(-1). These characteristics suggest the use of glucose oxidase from Aspergillus niger UAF-1 as an analytical reagent and in the design of biosensors for clinical, biochemical and diagnostic assays.

No MeSH data available.


Related in: MedlinePlus