D-hydantoinase

D-alanine DL-alanine hydantoin D-hydantoinase + d-A- carbamylamino acid amidohydrolas Firth, crystallopoietes ... 

An even more elegant approach for the production of D-phydroxyphenylglydne on an industrial scale uses foe bacterium. Agrobacterium radiobacter (Figure A8.8). The organism is able to produce both D-hydantoinase and a second enzyme, N-carbamoyl-D-amino acid aminohydrolase, which catalyse the hydrolysis of N-carbamoyl-D-amino add. 

Another approach for the synthesis of enantiopure amino acids or amino alcohols is the enantioselective enzyme-catalyzed hydrolysis of hydantoins. As discussed above, hydantoins are very easily racemized in weak alkaline solutions via keto enol tautomerism. Sugai et al. have reported the DKR of the hydantoin prepared from DL-phenylalanine. DKR took place smoothly by the use of D-hydantoinase at a pH of 9 employing a borate buffer (Figure 4.17) [42]. 

Figure 6.43 Dynamic kinetic resolution of (rac)-hydantoins by a D-hydantoinase.

D-p-Hydroxyphenylglycine is an important component of certain semi-synthetic antibiotics such as the semi-synthetic cephalosporins cefadroxil and cefatrizine and the semi-synthetic penicillin amoxicillin, with a combined world market in excess of 3 x 10 /a. Synthesis was possible from DL-5-monosubstituted hydantoins (cyclic ureides of amino acids) provided that a selective D-hydantoinase could be found, which would be competitive with chemical methods. 

Enzyme activity on a D (non-natural) configuration, non-protein cyclic amino acid derivative appears unlikely. However the D-hydantoinase reaction is very similar to the dihydroxypyrimidase present in pyrimidine metabolism. The original hydantoinase used was obtained from calf liver, but subsequently many active microorganisms were found, particularly a strain of B. brevis. The resulting D-A-carbamoyl amino acid can then be converted into product by treatment with nitrous acid (Figure 4.11). 

Figure 5.10 Agar overlay screening procedure to screen microbial populations for hydantoinase activity (Morin, Hummel and Kula, 1986). A screen for dihydropyiidinase activity based on Schiff base formation with PDMB (upper panel) led to the identification of numerous strains with D-hydantoinase activity, which in combination with a D-carbamoylase is employed to produce D-amino acids.

Morin, A. and Leblanc, D. (1998) Passive and active screening of D-hydantoinase-producing microorganisms. InTVew frontiers in screening for microbial catalysis, edited by K.Kieslich, C.J.van der Beek, J.de Bont and W.J.J. van den Tweel, pp. 133-142. Amsterdam Elseviers Science B.V. 

As evidenced by the well-established industrial production of D-amino acids (mostly D-phenylglycine and p-OH-D-phenylglycine by the hydantoinase/carbamoylase route), both D-hydantoinases and D-carbamoylases are well developed. Enantio-selectivity in most cases is not a problem (Gross, 1987). 

D-p-Hydroxyphenylglycine and its derivatives are important as side-chain precursors for semisynthetic penicillins and cepharosporines. Yamada and coworkers of our laboratory found that these amino acids can be efficiently prepared from the corresponding 5-monosubstituted hydantoins using the microbial enzyme D-hydantoinase [4]. 

The bacterial D-hydantoinase has been isolated as crystals from cells of Pseudomonas putida (= P. striata) (Table 1) [5]. Because the purified enzyme showed the highest activity and affinity toward dihydrouracil, the enzyme was identified as dihydropyrimidinase (EC. 3.5.2.2). Interestingly, the enzyme also attacked a variety of aliphatic and aromatic D-5-mono-substituted hydantoins, yielding the corresponding D-form of N-carbamoyl-a-amino acids. Thus, the enzyme can be used for the preparation of various D-amino acids. Under the conditions used for the enzymatic hydrolysis of hydantoin at pH 8 to 10, the L-isomers of the remaining hydantoins are racemized through base catalysis. Therefore, the racemic hydantoins can be converted quantitatively into N-carbamoyl-D-amino acids through this step. 

Decarbamoylation to D-amino acid was performed by treating the N-carbamoyl-D-amino acid with equimolar nitrite under acidic conditions [6]. But now, this step can also be carried out enzymatically. Recently, Shimizu and co-workers found a novel enzyme, D-decarbamoylase (IV-carbamoyl- n-amino acid amidohydrolase), which stereospecifically hydrolyzes JV-carbamoyl-D-amino acids, in several bacteria [7, 8], For example, Blastobacter sp. A17p-4 was found to produce D-decarbamoylase together with D-hydantoinase [8]. Therefore, a sequence of two enzyme-catalyzed reactions, the D-stereospecific hydrolysis of DL-5-(p-hydroxyphenyl) hydantoin and subsequent hydrolysis of the D-carbamoyl derivative to D-p-hydroxyphenylglycine, is possible (Fig. 1). Based on these results, a new commercial process for the production of D-p-hy-droxyphenylglycine has been developed [9]. 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Since D-hydantoinase was identified as dihydropyrimidinase, it is proposed that D-amino acid production from DL-5-monosubstituted hydantoins involves the action of the series of enzymes involved in the pyrimidine degradation pathway. Based on this proposal, D-decarbamoylase was thought to be identical with P-ureidopropionase (EC 3.5.1.6) which functions in pyrimidine metabolism. 

Hydantoinases and decarbamoylases have been applied for the production of optically active amino acids from DL-5-monosubstituted hydantoins. A variety of enzymes have been reported elsewhere. Runser et al. [33] reported the occurrence of D-hydantoinase without dihydropyrimidinase activity. Watabe et al. [34] reported that an ATP-dependent hydantoin-hydrolyzing enzyme is involved in the L-amino acid production from DL-5-monosubstituted hydantoin by Pseudomonas sp. NS671. This enzyme shows no stereospecificity. Hydan-toinase showing no stereospecificity and not requiring ATP was also reported [35]. Recently, hydantoin-racemizing enzymes were found [36,37], These enzymes make it possible to totally convert racemic substrates, which only slowly racemize under reaction conditions, to a single stereoisomer. The combinations of these hydantoin-transforming enzymes provide a variety of processes for optically active amino acid production (Fig. 4). 

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

While the production of D-amino acids is well established the preparation of L-amino acids is difficult due to the limited selectivity and narrow substrate spectrum of L-hydantoinases. This can be circumvented by employing rather un-selective hydantoinases in combination with very enantioselective L-carbamoyl-ases and carbamoyl racemases [90]. Furthermore, a D-hydantoinase has been genetically modified and converted into a L-hydantoinase. This enzyme can be used on a 100-kg scale for the production of L-tert-leucine [34]. Finally, the fact that the X-ray structure of an L-hydantoinase is known gives hope that side-directed mutagenesis will lead to improved L-hydantoinases [91]. 

One of the most widely used enzymatic methods for D-amino acid production is the hydantoinase process [4]. The great advantage of this process is that, potentially, any optically pure D-amino acid can be obtained using the corresponding substrate from a wide spectrum of D,L-5-monosubstituted hydantoins, which are readily accessible by chemical synthesis [5]. In this cascade of reachons the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme (D-hydantoinase). Further hydrolysis of the resulting N-carbamoyl D-amino acid to the free D-amino acid is catalyzed... 

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). 

---