Characterization of the biotin synthase reaction from Bacillus sphaericus using the photoreduced deazaflavin system

Using the photoreduced deazaflavin system, we examined the catalytic proper ties of biotin synthase of Bacillus sphaericus. In this assay system, only deazaflavin and fluorescent light replaced the complicated electron transpo rt system essential for biotin synthase. The enzyme activity obtained from this assay system was five-fold higher than that obtained in the assay syst em using a cell-free extract of the wild-type strain, B. sphaericus IFO3525 , as the electron transport system. Dithiothreitol (DTT) was essential for the enzyme reaction and the optimal concentration was 20 mM in the proposed assay system. Except for dithioerythritol, none of the other reductants te sted, such as 2-mercaptoethanol, glutathione, and sodium dithionite could e ffectively replace DTT. Among metal ions, only ferrous ion was effective an d the optimal concentration was 0.1 mM In this assay system. Compounds with two Or three phosphate groups, such as ATP, pyrophosphate and tripolyphosp hate had significant inhibitory effects. This observation could give one po ssible strategy for the enhancement of microbial biotin production.