Carbamoylases hydantoins

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

Recently, recombinant biocatalysts obtained using Escherichia coli cells were designed for this process. The overexpression of all enzymes required for the process, namely, hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771 was achieved. These cells were used for production of a-amino acids at the concentration of above 50 g 1 dry cell weight [37]. This is an excellent example presenting the power of biocatalysis with respect to classical catalysis, since a simultaneous use of three different biocatalysts originated from one microorganism can be easily achieved. 

In view of the last report, it is interesting that Wu et in Beijing have identified an organism, Sinorhizobium morekns S-5, that can convert the hydantoin of racemic -hydroxyphenylglycine into the D-amino acid. This, similar to the process just described, involves a hydantoinase and a carbamoylase, but both appear to be strictly D-specific. These authors again draw attention to the fact that under mildly alkaline conditions, spontaneous racemization of the hydantoin should permit a 100% conversion to the final D-product. 

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,... 

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]. 

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].

Monosubshtuted hydantoins are a-amino acids cyclically protected at both the carboxyl- and the a-amino group. They can be easily prepared from an aldehyde and isocyanate or by the Bucherer-Bergs synthesis and similar methods. Indeed, the hydantoin synthesis is also a prachcal method for the preparahon of the racemic amino acid. Enzymes belonging to the dihydro-pyrimidinase family hydrolyze hydantoins to the carbamoyl amino acid. The latter can be hydrolyzed in turn to the amino acid by a second enzyme, a carbamoylase. Both enzymes can discriminate between enantiomers and, if their action is cooperative, either the L- or the D-amino acid can be obtained (Scheme 13.10) [36]. What makes the system of special interest is that the proton in the 5-position of the hydantoin ring (it will become the a-hydrogen in the a-amino acid) is considerably more acidic than conventional protons in amino acid esters or amides and much more acidic than the amino acid itself. Thus, the hydantoin can be often racemized in situ at slightly basic pH where the enzymes are stiU stable and active. If these condihons are met. 

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. 

Very recently, however, a remarkably improved whole-cell biocatalyst coexpressing an L-carbamoylase, a hydantoin racemase, and an L-hydantoinase has been developed which showed a 50-fold higher productivity and is suitable for large-scale applications [17]. A prerequisite of this efficient whole-cell biocatalyst, which is applied at Degussa, was the successful inversion of the enantiospecificity of a previously D-selective hydantoinase by means of directed evolution [18]. These improvements have already been confirmed at a m3-scale using a batch reactor concept [17]. 

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 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. 

Natural cyclic amides such as 5,6-dihydrouracil, uracil and 5,6-dihydrothymine as well as hydantoin, 5-methylhydantoin and 5-hydroxymethylhydantoin are effective inducers for enzyme biosynthesis (for a more detailed review on induction experiments see reference13 ). In some cases, the dihydropyrimidinase (D-hydantoinase) is associated with an N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) and a hydantoin racemase1301. The previously proposed identity of the D-N-carbamoylase with the p-ureidopropionase (E. C. 3.5.1.6), which was assumed to be responsible for the hydrolysis of N-carbamoyl-P-alanine (see Fig. 12.4-7) 131-351 is no longer valid since the investigations of Ogawa et al. on different aerobic bacteria showed that the... 

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. ... 

Additionally, an Escherichia coli whole cell biocatalyst has been constructed containing the genes of hydantoinase, hydantoin racemase and i-N-carbamoylase from Arthrobacter aurescens in optimal proportions, so that during the reaction no l-N-carbamoyl amino acid occurs as an intermediate product any longer11431. 

Future work will show the impact of these methods on the biotechnological application of hydantoinases. Besides the applied research on hydantoinases for the production of amino acids, the natural functions and genetic organization of distinct hydantoinases, related hydantoin racemases and N-carbamoylases are still unknown and are of great interest for basic research. 

By directed evolution May et al.11541 succeeded in inverting the enantioselectivity of D-hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme. The improved hydantoinase also acquired a five-fold increase in activity. The recombinant E. coli cells expressing three heterologous genes (i. e. the evolved L-hydantoi-nase, L-N-carbamoylase, and hydantoin racemase) were found to produce 91 mM l-methionine from 100 mM D-5-(2-methylthioethyl)hydantoin in less than 2 h[1541. 

Enzymatic racemisation is an attractive option in DKR because the reactions catalysed by enzymes are performed under mild conditions. The Degussa group have recently described their successful commercialization of two DKR-based processes that employ racemases, namely (i) the DKR of 5-substituted hydantoins using whole cells coexpressing a L-carbamoylase, a hydantoin racemase and a hydantoinase and (ii) the DKR of N-acetyl amino acids using an acylase in combination with an N-acetyl amino acid racemase from Amycolatopsis orientalis. 

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