Single-pot enzymatic reaction sequence for the synthesis of D-glyceraldehyde-3-phosphate

A single-pot enzymatic reaction sequence has been designed for the synthesis of a key intermediate metabolite called D-glyceraldehyde-3-phosphate(D-GAP). The reaction sequence consistsof three enzymes and usesD-F16BP as a starting material (Fig.).The rabbit muscle aldolase (RAMA)-catalyzed reaction step is in favor of D-F16BP formation with an equilibrium constant of 10^–4M [1]. Therefore, the single-pot reaction sequence has been designed to shift the equilibrium and regenerate NADH. The capability of the reaction sequence was demonstrated by the enhanced equilibrium conversion from 1.4% to 96%. The reaction system was optimized in detail with regard to activity and stability of the enzymes and stability of the cofactors (NADH and NAD⁺) and D-GAP. Reaction kinetics models for each of the enzymes were formulated. The effect of co-substances on the activity ofthe enzymes was evaluated. The results elucidated that D-F16BP and sn-G3P do not influence the activity of FDH and HCOO^– does not affect the activity of RAMA, whereas D-F16BP and HCOO^–suppress the activity of sn-G3PDH. From the kinetics perspective and due to the stability of the enzymes, a continuous operation based on an enzyme membrane reactor was preferred