Enzyme Based Self-Prepared Kit to Measure the Glucose Concentration in Comparison with Standard Kit

In the present study, glucose oxidase was produced in shake culture by the parent Aspergillus niger with corn steep liquor as substrate. Glucose oxidase produced was then purified by subjecting to 60-85% saturation of ammonium sulfate precipitation and further purified by ion exchange and gel filtration chromatography respectively. Desalted sample of glucose oxidase showed the activity and specific activity of 39.19 U/ml and 9.07 U/mg. For ion exchange chromatography, 8 fraction of glucose oxidase had the maximum th activity of 19.99 U/ml with 17.70 U/mg specific activities. Gel filtration chromatography was applied by sephadex G-200 and glucose oxidase showed a specific activity of 54.243 U/mg. Peroxidase from horseradish obtained activity and specific activity of 21.78 U/ml and 9.64 U/mg, respectively after ammonium sulfate precipitation. DEAE-cellulose chromatography of peroxidase showed activity of 10.36 U/ml and specific activity 57.88 U/mg that was 5.07 U/ml and 66.71 U/mg after gel filtration. These purified enzymes were used to design 3 kits to estimate glucose with varying parameters. The self-prepared kit was compared with standard AMP kit. The self-prepared kit which gave closer result with the standard kit was kit A with enzyme concentrations of 2 ml glucose oxidase and 0.1 ml peroxidase at 546 nm wavelength in serum as well as in plasma.