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Enantiopure L-glyceraldehyde-3-phosphate (L-GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L-glyceraldehyde (L-GA) by ATP is used for the synthesis of L-GAP. L-GAP has a half-life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca , but not by L-GAP and inorganic orthophosphate. Moreover, equal amount of Ca exerts a different degree of inhibition relative to the activity without the addition of Ca depending on the Mg to ATP molar ratio. If the Mg to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred-tank reactor is the most efficient process for the synthesis of L-GAP.

A single-pot enzymatic reaction sequence has been designed for the synthesis of a key intermediate metabo-The reaction sequence consists of three enzymes and uses D-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 -4 M [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 of the 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.[1] M. D. Bednarski et al.,