Hydantoinases

Following epPCR and saturation mutagenesis at hot spots, the o-selective hydan-toinase from Arthrobacter sp. DSM 9771 was converted into L-selective variants. The best L-selective mutant showed a value of 20% ee at about 30% conversion, compared to the WT displaying ee = 40% in favor of the D-methionine-derivative. With the help of an appropriate L-carbamoylase, L-methionine itself was produced. This academic/ industrial effort provided several selective hydantoinases of industrial interest (O May, (Degussa-Hiils), personal communication, 2005). 

D-selective variants were also obtained 148). This academic/industrial collaboration led to the production of several highly selective hydantoinases of industrial interest 149). 

---

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 ... 

Several classes of enzymes have been used to separate stereoisomers of a-H-and a-disubstituted amino acids, eg amidases, nitrilases, hydantoinases, acylases and esterases. 

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. 

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. 

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)). 

Hsu s group in Taiwan have developed a procedure for the synthesis of (Y)-2-amino-4-phenylbutanoic acid, the phenylalanine homologue with one additional methylene group. Hydantoinase and L-A-carbamoylase genes have been cloned from different Bacillus species and overexpressed in E. coli. Both the R- and the 5-enantiomers were cleaved by the hydantoinase, but only the 5-form of the A-carbamoyl amino acid was hydrolyzed by the second enzyme. The reactions could be run in a single pot, with successive addition of the two enzymes, and were successful in the sense of giving a product of high chiral purity. However, the yield was... 

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. 

---