ABSTRACT

Along with other emerging health consequences, diabetes mellitus is one of the critical threats spreading all over the world with continuity. The investigation of glucose level in diabetic patients requires the key enzyme β-D-glucose:oxygen-1-oxidoreductase/glucose oxidase, where its optimized production by mutant derived strain could affect the economic burden and accuracy of the test. Wild type Aspergillus niger was subjected to ultraviolet radiation for enhanced production of glucose oxidase. It was found that UV-180-C is a potential mutant derived strain, screened by using 2-deoxy-Dglucose. Optimum production of glucose oxidase from A. niger UV-180-C was carried out by using CSL (2%), fermentation period (36 h), pH (5.5 and 4.5 for wild and mutant respectively), temperature (30°C), MgSO 4 .7H 2 O (0.0%), CaCO 3 (0.1%), KH 2 PO 4 (0.8 and 1.0% for wild and mutant strains), Urea (0.3%). Crude enzyme was subjected to ammonium sulfate precipitation and resulted into 145.8 UmL-1 activity. Glucose oxidase from mutant derived A. niger exhibited optimum pH at 6, temperature 20°C, K m 10mM and V max 142 UmL-1. The pyridoxal phosphate caused significant inhibition to the enzyme which indicates the presence of lysyl residues near or at the active site of the enzyme from both wild and mutant derived strains.

In the present study enhanced production of glucose aerodehydrogenase from Aspergillus niger has been achieved after optimizing the dose of chemical mutagen ethyl methane sulfonate (EMS) that has not been reported earlier. Different doses of mutagen were applied and a strain was developed basing upon the best production. The selected strain Aspergillus niger EMS-150-F was optimized for nutrient requirements in order to produce enzyme through fermentation and the results showed the best yield at 2% corn steep liquor (CSL), 36 hours fermentation time, pH 5, 30°C temperature, 0.3% KH2PO4, 0.3% urea and 0.06% CaCO3. The enzyme was then purified and resulted in 57.88 fold purification with 52.12% recovery. On kinetic characterization, the enzyme showed optimum activity at pH 6 and temperature 30°C. The Michaelis-Menton constants (Km, Vmax, Kcat and Kcat/Km) were 20 mM, 45.87 U mL−1, 1118.81 s−1 and 55.94 s−1 mM−1, respectively. The enzyme was found to be thermaly stable and the enthalpy and free energy showed an increase with increase in temperature and ΔS* was highly negative proving the enzyme from A. niger EMS-150-F resistant to temperature and showing a very little disorderliness.