Towards preparative asymmetric synthesis of beta-hydroxy-alpha-amino acids: L-allo-threonine formation from glycine and acetaldehyde using recombinant GlyA.

The diastereospecific formation of L-allo-threonine, catalyzed by the serine hydroxymethyltransferase GlyA form Escherichia coli, was studied with regard to the application in continuous processes. Process design will rely on a suitable description of enzyme stability and kinetics under relevant process conditions. Therefore, the effects of addition of organic co-solvents--methanol and acetonitrile--to the reaction mixtures on activity, stability, and diastereoselectivity were investigated. A series of progress curves from batch reactions at 35 degrees C in 50mM sodium phosphate buffer pH 6.6 and 50mM sodium phosphate buffer pH 6.6 in 20% methanol was used to estimate the respective kinetic parameters for an appropriate kinetic model. The experimental data agreed well with a kinetic model for an ordered reaction mechanism of the type bi-uni including the formation of a ternary complex and a pseudo-equilibrium assumption. The model was then applied in order to simulate the performance of the enzyme in an enzyme membrane reactor (EMR) and gave an excellent agreement with the corresponding experimental data. A space time yield of 227g L(-1)d(-1) was achieved in a continuous running EMR without significant loss of enzyme activity over 120 h of operation.