Thermal characterization of hyperproduced glucose oxidase from Aspergillus niger BCG-5 mutant strain

Abstract

Diabetes mellitus is a metabolic problem and is prevalent in many parts of the world. In developed countries, the incidence rate is 5% and an equal number is liable to develop the disease. One fundamental aspect of diabetes is an abnormality of glucose metabolism as due to insufficient action of insulin, owing either to its absence or to resistance in action. Blood glucose level in diabetes becomes so elevated that the glucose spills over into urine, providing a convenient diagnostic test for the disease. Three enzymes mutarotase, glucose oxidase and peroxidase are involved in the process of determination of glucose level. Glucose oxidase (GOD, beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) is an important enzyme, which catalyzes the oxidation of beta-D-glucose to D-glucono-1,5-lactone and hydrogen peroxide and finally to gluconic acid using molecular oxygen as electron acceptor. Aspergillus niger spore suspension of 48 hours Vogel's broth was exposed to gamma irradiation and 80 k Rad. dose was selected as optimized dose to induce the mutation. For selection of resistance to catabolite repression, 2-deoxy-D-glucose was used at 1 mg mL-1. An intracellular glucose oxidase was produced from the mycelium extract of a gamma rays mutated strain of Aspergillus niger BCG-5. Furthermore, enzyme was purified and subjected to kinetic/thermodynamic characterization. The enzyme was purified to a yield of 61.91%, 255.23 fold purification and specific activity of 3910.06 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 28 mM and Vmax 60 U mL-1 with a molecular weight of 132 kDa. The enzyme exhibited optimum catalytic activity at pH 5. Temperature optimum for glucose oxidase catalyzed D-glucose oxidation was 40degC. The enzyme showed a high thermostability having a half-life 12.16 minutes, enthalpy of denaturation 0.171 kJ mol-1 and free energy of denaturation 77.64 kJ mol-1. Glucose oxidase, produced/purified from the mycelium extract of Aspergillus niger BCG-5 exhibited high catalytic properties and thermostability. Breakthrough work to be presented: These characteristics suggest the use of glucose oxidase from mutant Aspergillus niger BCG-5 as a valuable analytical tool and in the design of biosensors for clinical, biochemical and diagnostic assays.