Racemases hydantoin racemase

Figure 12.4 Multiple alignment of the amino acid sequences of hydantoin racemases. Hydantoin racemase 1 from A tumefaciens CSS (AtHyuAl), CenBank accession no. AY436503 hydantoin racemase from S. meliloti CECT 4114 (SmeHyuA), CenBank accession no. AY393697 hydantoin racemase 2 from A tumefaciens CSS (AtHyuA2),...

Glu racemase Asp racemase Hydantoin racemase Maleate isomerase AMDase... 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

Racemic hydantoins result from the reaction of carbonyl compounds with potassium cyanide and ammonium carbonate or the reaction of the corresponding cyanohydrins with ammonium carbonate (Bucherer-Bergs reaction). Hydantoins racemize readily under basic conditions or in the presence of hydantoin racemase, thus allowing DKR (Figure 6.43). Hydantoinases (EC 3.5.2.2), either isolated enzymes or whole microorganisms, catalyze the hydrolysis of five-substituted... 

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. 

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,... 

Figure 7.14 Hydantoinase/carbamolyase/(hydantoin racemase) process to d- or L-amino acids. 

M. Pietzsch, C. Syldatk, and F. Wagner, Isolation and characterization of a new, non pyridoxal-5 -phosphate dependent hydantoin racemase, DECHEMA Biotechnol. Conf. 1990, 4, 259-262. 

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]. 

In elegant work the enantioselectivity of a hydantoinase from Arthrobader species for the production of l-methionine in Escherichia coli has been inverted [87]. The approach is similar to the one used in the evolution of (S)- and ( -selective lipases (see above). All known hydantoinases are selective for D-5-(2-methylthioethyl)hydantoin (d-18) which leads to the accumulation of N-carbamoyl-D-methionine (d-19), conversion being complete if the conditions of dynamic kinetic resolution are upheld [88], in this case by the use of a racemase or pH >8 (Fig. 11.22). 

Hydantoin Racemase the Key Enzyme for the Production of Optically Pure oc-Amino Acids... 

Given the important role that hydantoin racemase plays in the production of optically pure D-amino acids, allowing the racemization of 5-monosubstituted... 

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].

CenBank accession no. AY436S04 hydantoin racemase from A. aurescens DSM 3747 (AauHyuA), CenBank accession no. AF146701 hydantoin racemase from M. liquefaciens A) 3912 (MliHyuA), CenBank accession no. BD1S1023 hydantoin racemase from Pseudomonas sp. NS671 (PspHyuE), CenBank accession no. M84731. 

Figure 12.5 Phylogenetic analysis of the amino acid sequences of hydantoin racemases characterized to date. For abbreviations of the enzymes, see the caption to Figure 12.4.

Although on the whole the hydantoin racemases have shown high thermal stability, with optimal activity at 55 °C (Table 12.2), the optimal temperatures for the ones from Pseudomonas and Sinorhizobium decrease to 45 and 40 °C, respectively. However, the optimal pH is higher than 8, except for both hydantoin racemases from Agrobacterium. This low alkaline pH avoids chemical racemization. Consequently, the racemization of the d- or L-5-monosubstituted hydantoins in an industrial process will only occur enzymatically. 

The activity of the purified hydantoin racemase enzymes has been assayed in the presence of different metal ions, the chelating agent EDTA, and the reducing compound dithiothreitol, in order to study whether they are metaUoenzymes. Most... 

Table 12.2 Biochemical properties of hydantoin racemases described to date.

Substrate EnantioselectMty and Kinetic Analysis of Hydantoin Racemases 1181... 

Figure 12.7 Reaction profile of the enzymatic racemization of D- and L-5-monosubstituted hydantoin to the racemic mixture by hydantoin racemase enzyme monitored by chiral HPLC (see the methodology in [28]).

Figure 12.8 Enzymatic racemization of the D-isomer ( ) and L-isomer (O) of (a) 5-benzylhydantoin and (b) 5-ethylhydantoin by hydantoin racemase 1 from A. tumefaciens C58 (AtHyuAl).

Table 1Z3 Kinetic parameters of A. tumefaciens C58 hydantoin racemase 1 and 2 (ATHyuAI and AtHyuA2).

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