MONITORING THE ACTIVITY OF GLUCOSE-OXIDASE DURING THE CULTIVATION OF ASPERGILLUS-NIGER USING NOVEL AMPEROMETRIC SENSOR WITH 1,1'-DIMETHYLFERRICINIUM AS A MEDIATOR

1, 1'-dimethylferricinium (DMF+), a deep blue, and stable mediator, wa s prepared from a water-soluble 1, lferrocene(DMF):2-hydroxyprophyl-be ta-cyclodextrin complex via enzymatic oxidation using immobilised bili rubin oxidase. This mediator was superior to other soluble ferrocenes, notably carboxyferrocene, in terms of both solubility (110 mM vs 0.5 mM) and oxidation potential (150 mV vs 300 mV against Ag/AgCl). Althou gh the cyclic voltammogram of DMF+ was electrochemically equivalent to DMF, the use of the former resulted in a significantly lower backgrou nd current (< 10 nA vs 30 nA). Because of its higher solubility, conce ntrated stock solutions of DMF+ can be prepared and supplied to the el ectrode. This is of particular importance when the signal is severely limited by the rate at which the working electrode can oxidise DMF to DMF+. A linear response of current versus units of glucose oxidase (GO D) was obtained up to 0.5 unit/ml. The detection limit was estimated t o be 0.03 unit/ml and the response time was 2.5 min or less. The amper ometric system was used successfully to follow the GOD activity during the growth of Aspergillus niger a well-known GOD producer. The result s obtained correlated well with a standard absorbance-based assay usin g dichlorophenol-indophenol (DCPIP). The K(M) of GOD for the glucose i n the lysate was measured as 38 mM. A reduced response and higher K(M) (48 mM) of the cell homogenate, compared to the lysate, illustrated t he requirement for the DMF+ and glucose to diffuse across the cell mem brane to interact with GOD in whole cells.