As reported previously, enzymatic production of ATP from adenine by resting cells of Brevibacterium ammoniagenes (Fujio and Furuya 1983) accumulated 13.0 mg of ATP · Na2 · 3H2O/ml, but ATP formation ceased within 6–8 h. Simultaneous addition of magnesium ion and phytic acid, a chelator of divalent cations, allowed ATP formation to continue longer, and 24.2 mg of ATP · Na2 · 3H2O/ml was accumulated in 10 h. However, ATP formation ceased thereafter.
This second cessation was found to be caused by the lack of magnesium ion active as a co-factor (Mgact). The Mgact was tentatively taken as the difference between soluble magnesium ion (Mgsol) and the ion chelated by an equimolar amount of ATP (MgATP), namely Mgact=Mgsol-MgATP. In order to provide Mgact, sufficient phytic acid had to be added at the beginning of the reaction and magnesium ion was also added intermittently. Under these conditions ATP formation continued further, and the rate of ATP formation was increased; 37.0 mg of ATP · Na2 · 3H2O/ml was accumulated in 13 h.
Since whole culture broth is preferable to frozen cells as a practical enzyme source, the conditions neccessary for use of whole culture broth of B. ammoniagenes were also investigated.