19-08-2014, 11:30 AM
Glucose oxidase ( -D-glucose: oxygen-1-oxidoreductase) is a flavoprotein, which catalyzes the oxidation of -D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with the simultaneous production of hydrogen peroxide. The hydrogen peroxide formed is used to oxidize a chromogenic substrate (o-dianisidine) in a secondary reaction with horseradish peroxidase with a resultant colour change.
Glucose oxidase ( -D-glucose: oxygen-1-oxidoreductase) is a flavoprotein, which catalyzes the oxidation of -D-glucose to gluconic acid, by utilizing molecular oxygen as an electron acceptor with the simultaneous production of hydrogen peroxide. The hydrogen peroxide formed is used to oxidize a chromogenic substrate (o-dianisidine) in a secondary reaction with horseradish peroxidase with a resultant colour change. Glucose oxidase (GOD) can be used for removal of oxygen and glucose from various materials to prevent undesired reactions. Use of GOD in biosensors is the major application in industrial as well as in diagnostic field now a day.
GOD was produced by submerged fermentation with Aspergillus niger NCIM 545. Optimization of media components and conditions such as carbon, nitrogen, initial pH, temprature, innoculum size and effect of CaCO3 were done by conventional one-factor-at-a-time method. Plackett- Burman design was used to screen the most significant fermentation parameters affecting glucose oxidase production.
CaCO3, peptone and MgSO4 were found to be most significant among the six variables screened.
Central Composite Design (CCD) was applied to variables obtained from Plackett–Burman design for optimizing the concentrations and interactions among them. After statistical optimization 7 fold increase in activity was observed as compared to media optimized with one-factor-at-a-time method. -aminopropyltriethoxysilane & polyethyleneimine were used for the activation of glass surface for covalent immobilization of GOD. Immobilization with polyethyleneimine as an activator was found to be more effective. When the surface of glass beads became rough, they showed higher enzyme binding to the glass (about 70%). Near about all the enzyme which gets bound to glass surface was found to be active and very negligible loss of enzyme was seen with this method. Effect of various variables which are affecting immobilization of GOD such as GOD to catalase ratio, polyethyleneimine concentration and gluteradehyde concentration were optimized using CCD design.