A variety of optically pure proteinogenic and non-proteinogenic amino acids can be synthesized using the „hydantoinase method" [1].Two enzymes are involved in the hydrolysis of D,L-5-monosubstituted hydantoines and N-carbamoylamino acids. The hydantoinase is not stereospecific for a range of interesting products [2] so that the key step in these reactions is represented by the N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase). Both enzymes are unstable under typical reaction conditions and therefore not reusable. This holds also for resting cell biotransformations. The primary goal of this work was the immobilization of both enzymes in order to obtain higher catalyst stability for process improvement.

In a previous contribution [3] we were able to show that both enzymes could be significantly stabilized by immobilization onto various supports but the obtained coupling yields as well as specific activities remained low. In order to optimize the immobilization procedure the influence of the protein concentration was investigated. We found a dependance between protein concentration, activity yield and specific actvity in such a way that the yield decreases at high protein concentrations while the specific activity is found to increase. By the use of a recombinant carbamoylase from E. coli [4] the specific activity of the immobilisate could significantly be improved.

Current investigations with the purified recombinant enzymes are focusing on the immobilization of a recombinant hydantoinase with regard to higher specific activities of the immobilizate. Systematic investigations of adsorption processes for different supports are intended since an activity loss of about 80% was found in the case of carbamoylase adsorption.

[1] Pietzsch, M. and Syldatk, C. (1995), in Enzyme Catalysis in Organic Synthesis (Drauz, K. and Waldmann, H., eds.), VCH Verlag, Weinheim, Germany, 409-431

[2] May, O. (1998), J. Biotechnol., 61, 1-13.

[3] Pietzsch, M., Oberreuter, H., Petrovska, B., Ragnitz, K., Syldatk, C. (1998), in Stability and Stabilization of Biocatalysts, Progress in Biotechnology 15 (Ballesteros, A., Plou, F. J., Iborra, J. L., Halling, P. J., eds), Elsevier, Amsterdam, 517-522

[4] Wilms, B., Wiese, A., Syldatk, C., Mattes, R., Altenbuchner, J., and Pietzsch, M. (1999), J. Biotechnol., 68(2-3), 101-113