Enzymic routes to dihydroxyacetone phosphate or immediate precursors

Dihydroxyacetone phosphate (DHAP) is an important substrate for enzymic syntheses of saccharides by means of dihydroxyacetone phosphate aldolases. A convenient precursor of DHAP is l-glycerol-3-phosphate (l-G3P), the enzymic synthesis of which may be achieved by phosphate transfer owing to an ATP-regenerating system. The phosphorylation of both glycerol and dihydroxyacetone by means of an ATP-regenerating system based on phosphoenolpyruvate, pyruvate kinase and glycerol kinase is examined in order to optimize the reaction conditions. The composition of the reaction mixture should not exceed concentrations of 300 mM of each phosphoenolpyruvate and glycerol. Otherwise, the productivity would be affected by substrate inhibition. An operating temperature of 50 °C may be applied, when the enzymes should be used only once. At a temperature of 35 °C, a high half-life of both enzymes glycerol kinase and pyruvate kinase is achieved. Various concentrations of the enzymes are tested to optimize the use of the enzymatic activity. A ratio of 2:3 of pyruvate kinase and glycerol kinase activity seems to be adequate. Further studies show that 10 mM ATP is enough to maintain a permanent supply of the cofactor. It is shown, how DHAP may be obtained from l-G3P by means of an l-G3P oxidase and catalase, the latter being used not only to decompose the hydrogen peroxide formed during the course of reaction, but also to supply the reaction system with oxygen. This is achieved by feeding a H2O2 solution in a way to keep the oxygen level constant in the reaction mixture.