| Summary: | Protein immobilization is proving to be an environmentally friendly strategy for manufacturing biochemicals at high yields and low production costs. This work describes the optimization of the so-called “double-racemase hydantoinase process,” a system of four enzymes used to produce optically pure l-amino acids from a racemic mixture of hydantoins. The four proteins were immobilized separately, and, based on their specific activity, the optimal whole relation was determined. The first enzyme, d,l-hydantoinase, preferably hydrolyzes d-hydantoins from d,l-hydantoins to N-carbamoyl-d-amino acids. The remaining l-hydantoins are racemized by the second enzyme, hydantoin racemase, and continue supplying substrate d-hydantoins to the first enzyme. N-carbamoyl-d-amino acid is racemized in turn to N-carbamoyl-l-amino acid by the third enzyme, carbamoyl racemase. Finally, the N-carbamoyl-l-amino acid is transformed to l-amino acid by the fourth enzyme, l-carbamoylase. Therefore, the product of one enzyme is the substrate of another. Perfect coordination of the four activities is necessary to avoid the accumulation of reaction intermediates and to achieve an adequate rate for commercial purposes. The system has shown a broad pH optimum of 7–9, with a maximum activity at 8 and an optimal temperature of 60 °C. Comparison of the immobilized system with the free protein system showed that the reaction velocity increased for the production of norvaline, norleucine, ABA, and homophenylalanine, while it decreased for l-valine and remained unchanged for l-methionine.
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