New Developments in the Chemo-Enzymatic Production of Amino Acids J. Kamphuis, W. H. J. Boesten, Q. B. Broxterman, H. F. M. Hermes, J. A. M. van Balken, E. M. Meijer, and H. E. Schoemaker DSM Research, Bio-organic Chemistry Section, P.O. Box 18, 6160 MD Geleen, The Netherlands Dedicated to Professor Armin Fiechter on the occasion of his 65th birthday Recent progress in the chemo-enzymatic production ,of amino acids is reviewed. Both recently developed commercial processes and potentially important new developments are discussed. Emphasis is placed on the use of acylases, aminopeptidases and hydantoinases. The discovery of D-specific enzymes in combination with racemases is an exciting and promising new area. Also, a goal-orientated approach towards the selective generation of these novel enzyme activities using in vivo protein engineering techniques is highlighted. The interest in dipeptide sweeteners hastriggered a major research effort towards the production of L-phenylalanine and D-alanine. A number of methods for the production of these amino acids is briefly discussed. Finally, chemo-enzymatic methods for the synthesis of enantiomerically pure c-alkyl-c-amino acids are reviewed. 1 General Introduction . 134 2 Enzymatic Resolution Processes for the Production of -H-Amino Acids . 136 2.1 Applications of Amino Acids . 136 2.2 Acylases . 136 2.3 Amidases/Aminopeptidases . 142 2.4 Hydantoinases/Decarbamoylases . 146 2.5 New Developments . 147 2.5.1 Microbial Hydrolysis of Amino Acid Carbamates . 147 2.5.2 Reversal of Stereoselectivity . 149 2.5.3 Racemases . : . 156 3 Methodologies to Obtain Novel Enzyme Activities . 162 4 Approaches to the Chemo-Enzymatic Synthesis of Amino Acids Used in the Production of 167 Artificial Dipeptide Sweeteners . 167 4.1 L-Phenylalanine . . 169 4.2 D-Alanine . 5 Chemo-Enzymatic Synthesis of -Alkyl-ct-Amino Acids . 171 6 Conclusions and Future Prospects . 182 7 References . 182 Advances in Biochemical Engineering/ Biotechnology, Vol. 42 Managing Editor: A. Fiechter ?9 Springer-Verlag Berlin Heidelberg 1990 134 J. Kamphuis et al. 1 General Introduction From time immemorial, amino acids have played an important role in biology, biochemistry and (industrial) chemistry. Amino acids are the building blocks of proteins and they play an essential role in the regulation of the metabolism of living organisms. The interest in the commercial production of amino acids dates back to 1908 with the discovery of monosodium glutamate (MSG) as a seasoning agent by Kikunae Ikeda (see 1). At first MSG was industrially obtained from wheat, soybean and other plant proteins. Since 1957, however, microbial production processes have been successfully developed for glutamic acid and subsequently also for other proteinogenic amino acids like lysine, valine, proiine, threonine, phenylalanine etc. However, a detailed discussion of these microbial processes and the application of novel genetic engineering techniques for strain improvement would go beyond the scope of the present review. In general, from a commercial viewpoint it can be stated that when comparing chemo-enzymatic methods with microbial processes, the latter are highly competitive for the large-scale production (thousands of tons per year) of naturally occurring proteinogenic L-amino acids, whilst for the production of natural amino acids on a small scale or for the pro- duction of synthetic D- and/or L-amino acids, chemo-enzymatic methods are the methods of choice in addition to all-chemical procedures. Current interest in developing peptide-derived chemotherapeutics has heigh- tened the importance of rare and non-proteinogenic enantiomerically pure amino acids. For example, D-phenylglycine and D-p-hydroxy-phenylglycine are building blocks for the broad spectrum 13-1actam antibiotics ampicillin and amoxycillin, respectively. The natural amino acid L-valine is used as feedstock in the microbial production of the cyclic undecapeptide cyclosporin A, which has immuno-sup- pressive activity and is used in human transplant surgery. Also, amino acids are versatil