Hydantoinase-carbamoylase system

The Hydantoinase-Carbamoylase System for o-Amino Acid Synthesis 5-... 

Scheme 13.13 Hydantoinase-Carbamoylase system for the deracemization of a-amino acids via DKR with in situ...

Hydantoinase-Carbamoylase System for t-Amino Acid Synthesis Despite a number of reports of strains with L-selechve hydantoin-hydrolyzing enzymes [38] the commercial application of the hydantoinase process is stiU restricted to the production of D-amino acids. Processes for the production of L-amino acids are Umited by low space-time yields and high biocatalyst costs. Recently, a new generation of an L-hydantoinase process was developed based on a tailor-made recombinant whole cell biocatalyst. Further reduction of biocatalyst cost by use of recombinant Escherichia coli cells overexpressing hydantoinase, carbamoylase, and hydantoin racemase from Arthrohacter sp. DSM 9771 were achieved. To improve the hydan-toin-converting pathway, the level of expression of the different genes was balanced on the basis of their specific activities. The system has been appUed to the preparation of L-methionine the space-time yield is however still Umited [39]. Improvements in the deracemization process from rac-5-substituted hydantoins to L-amino acids still requires a more selective L-hydantoinase. 

Figure 12.3 Reaction profile of D-methionine production from D,L-methylthioethylhydantoin (d.l-MTEH) using (a) a double system with D-hydantoinase and D-carbamoylase enzymes and (b) a triple system with hydantoin racemase enzyme as third enzyme. Symbols , D-methionine O, N-carbamoyl d-methionine , D,L-methylthioethylhydantoin , sum of all three [9].

The use of the whole cell system is necessary due to the limited stability of the carbamoyl-hydrolyzing enzyme and to the different requirements for optimal reactivity. Best carbamoylase activity is obtained by lowering the reaction pH slightly, which is regulated from the development of carbon dioxide from the first reaction step. The D-hydantoinase-D-carbamoylase process has proved successful for the preparation of a large number of D-amino acids [37] (Scheme 13.13). 

The other major class of cyclic amino acid derivative used in dynamic resolution reactions is the hydantoin group. Like oxazolinones, hydantoins readily undergo racemisation under mild conditions. Systems involving a two step procedure using D-hydantoinase and a carbamoylase were reported to provide a route to D-amino acids[S2, 53). Dynamic resolution of a p-hydroxyphenyl substituted hydantoin was reported in 1987[541. Using the intact cells of Pseudomonas sp. AJ-11220, the amino acid was prepared in over 90% yield, as shown in Fig. 9-24. This hydrolytic procedure leads directly to the amino acid, and the same enantiomer of product, the D-amino acid, was obtained independently of the stereochemistry of the substrate. 

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