D,L-hydantoins

Chemically synthesised D,L-hydantoins prepared from the corresponding aldehydes via die Bucherer Berg reaction are converted by the bacterial cells (Bacillus brevis), containing a D-spedfic hydantoinase, to a mixture of D-N-carbamoyl amino acid and L-hydantoin. The latter compound undergoes rapid and spontaneous racemisation under the conditions of the reaction, therefore, in principle 100% of the hydantoin is converted into the D-N-carbamoyl compound. The D-amino add is obtained after treatment of the D-N-carbamoyl compound with nitrous add. This process is operated on an industrial scale by the Japanese firm Kanegafuchi. 

D,L-hydantoin W-carbamoyl-D-amino acid D-amino acid... 

Dihydiopyiiinidinase- (hydantoinase-) based processes have been successfully employed for the production of D-amino adds, particularly D-p-hydroxyphenylglydne (7,25,77,78). D,L-hydantoins are chemically synthesized from the corresponding aldehydes using the Bucherer-Berg... 

All microorganisms producing D-aminoacylases commonly produce L-aminoacy-lases as well. Therefore, to reach high optical purity of the D-amino acids produced from the respective N-acetyl-D,L-amino acids, the D-aminoacylases have to be separated from the L-aminoacylases (Table 12.3-13). However, this is a disadvantage in view of an industrial application since additional purification steps lead to more expensive enzymes and thus add costs to the whole production process. This is one of several reasons why it is widely accepted today that the production of D-amino acids by enzyme-catalyzed hydrolysis of D,L-hydantoins seems to be more promising than the D-aminoacylase route via N-acetyl-D,L-amino acids. The enzyme-catalyzed synthesis of D-amino acids from the respective D,L-hydantoins is described in Chapter 12.4. 

Figure 17-15. Enzymatic synthesis of d- or L-amino acids from 5-substituted D,L-hydantoins through N-carbamoyl-D- or L-amino acids.

Using epPCR followed by saturation mutagenesis at hot spots, the D-selective hydantoinase from Arthrobacter sp. DSM 9771 was converted into an L-selective variant showing a fivefold increase in activity. Whole E. coli cells expressing the evolved L-hydantoinase and a hydantoin racemase led to the production of 91 mM L-methionine from 100 mM of d,l-MTEH as starting substrate. The best L-selective mutant showed an ee value of 20% at about 30% conversion, compared to the wild type displaying ee — 40% in favor of the D-methionine derivative. With the help of an appropriate L-carbamoylase, L-methionine itself was produced. In the project,... 

Syldatk, C., Laufer, A., Muller, R. and Hobe, H. (1990) Prodnction of optically pure D and L-df-amino acids by bioconversion of D,L-5-monosnbstituted hydantoin derivatives. Advances in Biochemical Engineering, 41, 28-75. 

Gross, C., Syldatk, C. and Wagner, F. (1987) Screening method for micro-organisms producing L-amino acids from D,L-5-monosubstituted hydantoins. Biotechn. Techniques, I, 85-90. 

T. Wagner, B. Hantke, and F. Wagner, Production of L-methionine from d,l-5-(2-ethylthioethyl) hydantoin by resting cells of a new mutant strain of Arthrobacter species DSM 7330, J. Biotechnol. 1996, 46, 63-68. 

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... 

High velocities of chemical racemization have only been observed for d,l-5-phenyl- and D,L-5-p-hydroxy-phenylhydantoin, because of the resonance stabilization by the 5-subshtuent, while all other hydantoins take many hours to racemize... 

Figure 12.2 Reaction scheme for the hydrolysis of d,l-5-monosubstituted hydantoin derivatives to the corresponding D-amino acids.

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 development of this multienzymatic system for the production of D-amino acids from any D,L-5-monosubstituted hydantoin allows the hydantoinase process to produce not only two amino acids, such as D-phenylglycine and D-p-hydroxy-phenylglycine (as explained at the beginning of the chapter), but also many non-natural D-amino acids that could be components of potential pharmaceuticals. 

The reaction concept with this new hydantoinase-based biocatalyst is economically highly attractive since it represents a dynamic kinetic resolution process converting a racemic hydantoin (theoretically) quantitatively into the enantiomerically pure L-enantiomer [19]. The L-hydantoinase and subsequently the L-carbamoylase hydrolyze the L-hydantoin, l-11, enantioselectively forming the desired L-amino acid, l-2. In addition, the presence of a racemase guarantees a sufficient racemiza-tion of the remaining D-hydantoin, d-11. Thus, a quantitative one-pot conversion of a racemic hydantoin into the desired optically active a-amino acid is achieved. The basic principles of this biocatalytic process in which three enzymes (hydan-toinase, carbamoylase, and racemase) are integrated is shown schematically in Fig. 9. 

The six-membered ring systems 5,6-dihydropyrimidine, 5,6-dihydrouracil and 5,6-dihydrothymine can be hydrolyzed by the enzyme dihydropyrimidinase (E.C. 3.5.2.2), which is involved in the degradation of pyrimidine nucleotides. This widely spread, inducible catabolic enzyme is strictly D-selective in contrast to the L-selective dihydroorotase (E. C. 3.5.2.3), which is involved in the opposite anabolic pathway (see above). Another name often used in the literature for the dihydropyrimidinase is d-hydantoinase, because it is also able to hydrolyze D,L-5-monosubstituted hydantoin derivatives with high activity. Both reactions are shown in Fig. 12.4-7. 

Nishida et al. 46, Syldatk et al. 47, 48, Yamashiro et al. 49, 50, and Yokozeki et al. 51-53 found new L-5-arylalkylhydantoinases and a N-carbamoyl-L-amino acid amidohydrolases (L-N-carbamoylase), which are involved in the L-selective cleavage of 5-arylalkylhydantoins and could be most favorably induced by D,L-5-indolylme-thylhydantoin or its N-3-methylated derivative17. The natural functions of these enzymes are not yet known, while one of the associated N-carbamoyl-L-amino acid amidohydrolases (L-N-carbamoylase) was also shown by Syldatk et al. to be reactive on N-formyl-L-amino acids[54]. In this strain both, hydantoinase and L-N-carbamoylase were shown to occur in combination with a hydantoin racemase 7, 55, 56. Resting cells were used for the industrial production of L-amino adds from d,l-5-monosubstituted hydantoin derivatives as shown in Fig. 12.4-2 57. ... 

Inductor dihydrouracil, hydantoin N-3-Methyl-D,L-5-indo- Hydantoin 5-cyanoethyl-hydantoin... 

Yokozeki et al.151-531 and Syldatk et al.17, 1251 as well as in Flavobacterium sp. by Nishida et al.1461. These so called L-5-arylalkylhydantoinases have comparable substrate specificities and are especially active towards the hydrolysis of hydantoin derivatives with aromatic substituents, as can be seen from Fig. 12.4-16. They could only be induced by D,L-5-indolylmethylhydantoin or the corresponding N-3-methyl derivative of a variety of hydantoins and natural cyclic amides17, S3, 124, 125l... 

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