The further optimization of 3-ketodisaccharide formation with sucrose, leucrose and iso maltulose was studied with special regard to pH and oxygen concentration in the reaction mixture with resting cells of Agrobacterium tumefaciens. It was found that the optimal pH values for the highest reaction rate and highest yield were different as the pH affected the stability of the 3-keto derivatives formed. A pH shift to 5.0 clearly reduced the enzymatic degradation of the 3-keto derivatives thus stabilizing them. The influence of constant oxygen concentrations on 3-ketosucrose formation was tested showing results not explicable with normal Michaelis-Menten kinetics. For each substrate a maximum of reaction rate and yield were obtained at very low oxygen concentrations.