Hydantoinases hydantoins

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. 

Both pure L- and D-amino acids can be made using hydantoinase enzymes. These enzymes catalyze the stereoselective hydrolysis of racemic hydantoins such as (50) which is used for the production of D-alanine (15) (58). 

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

N-carbamyl-D-phenylglyc DL-5-phenylglycine hydantoin hydantoinase Pseud, putida ... 

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. 

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]. 

Figure 2.13 Reactions and enzymes involved in the production of L-amino acids from racemic hydantoins by the three-enzyme hydantoinase process [55],...

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]. 

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... 

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

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. 

Kanegafuchi Chemical Industries produce D-p-hydroxyphenyl glycine, which is a key raw material for the semisynthetic penicillins ampicillin and amoxycillin. Here, an enantioselective hydantoinase is applied to convert the hydantoin to the D-p-hydroxyphenyl glycine. The quantitative conversion of the amide hydrolysis is achieved because of the in situ racemization of the unreacted hydantoins. Under the conditions of enzymatic hydrolysis, the starting material readily racemizes. Therefore, this process enables the stereospecific preparation of various amino acids at a conversion of 100% [38]. 

It has been shown recently that papain exhibits hydantoinase activity. This enzyme of plant origin hydrolyzes not only 5-monosubstituted but also 5,5-disubstituted hydantoins to the corresponding N-carbamoylamino acids. Since chemical hydrolysis of the latter yields the corresponding amino acids, this approach may be of interest in amino acid synthesis [145],... 

The two groups of enzymes discussed here have attracted attention because both offer a useful broad spectrum of substrate specificity. They are grouped together because in the context of amino acid synthesis they form a natural pair. Amino acid hydantoins are convenient from the standpoint of organic synthesis. The hydantoinases cleave the ring, producing the A-carbamoyl derivative of the amino acid. This must then be further hydrolyzed to obtain the free amino acid, and this step is likely to be strictly enantioselective (Equation (10)). 

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. 

This enzyme [EC 3.5.2.2], also called hydantoinase, catalyzes the hydrolysis of 5,6-dihydrouracil to produce 3-ureidopropionate. The enzyme can also utiUze dihydrothymine and hydantoin as substrates. 

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,... 

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. 

The hydantoinase process, consisting of a racemization reaction and hydrolyses of the hydantoin and the carbamoylic acid (Figure 7.14), has been enjoying much industrial success (> 1000 tpy) for almost 30 years in the production of D-amino acids such as D-phenylglycine and p-OH-phenylglycine which serve as side chains for /3-lactam antibiotics ampicillin and amoxicilin (Cecere, 1976). 

In contrast to acyl amino acids (pKa > 30) or amides, most 5-monosubstituted hydantoins racemize comparatively easily phenyl-substituted ones even racemize spontaneously at slightly alkaline conditions as their pK.d is around 8 (Kato, 1987). Under spontaneous or enzymatic racemization (Pietzsch, 1990), racemic hydantoins with the help of enantioselective d- or L-hydantoinases and the respective carb-... 

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

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. 

Fig. 2. Reactions catalyzed by hydantoin-hydrolyzing enzymes (hydantoinases)...

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). 

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