Hydantoinase process

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

Several other companies have developed patented processes to D-hydroxyphenylglycine. These include Bayer, DSM, SNAM-Progetti and Ajinomoto. DSM has got access to several technologies and is now carrying out the hydantoinase process at DSM Deretil in Spain. 

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). 

Scheme 6.25 The hydantoinase process for the production of amino acids.

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... 

Consequently, although the hydantoinase process has been successfully used for producing 100% D-phenyl- and D-p-hydroxy-phenylglycine [9], only 50% of the... 

Once several hydantoin racemases had been studied and characterized, their behavior, together with the other enzymes involved in the hydantoinase process. 

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. 

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. 

D-Phenylglycine and D-p-OH-phenylglycine are important side chain moieties in the synthesis of semisynthetic penicillins and are produced in several thousand tons per year using the hydantoinase process 3, 1001. The different methods that this... 

Figure 12.4-12. Industrial production of D-4-hydroxyphenylglycine acids by the D-hydantoinase process.

Resting cell L-hydantoinase processes were first developed for the industrial production of L-tryptophan by the companies Ajinomoto and Tanabe[46- 51-53f in 1992 the Ruttgers company tried to enter the amino acid marked with a resting cell... 

Some of the current reports on hydantoinase processes focus on isolation and the recombinant expression of thermostable enzymes[84, 86, 87, 95, 151. Processes at an elevated temperature would increase the solubility and racemization rate of hydantoins. Therefore, the increased thermostability of these enzymes is very useful, if the specific activities are still high. 

Here the biotransformation competes with the classical chemical route (Fig. 19-29), which employs bromocamphorsulfonic acid (Br-CAS) as the resolving agent. In both routes phenol is used as raw material since p-hydroxybenzaldehyde is too expensive. The hydantoinase process for phenylglycines does not necessarily need an extra racemization step since the hydantoin is racemized in situ at an alkaline pH. Because of the dynamic resolution in the case of this biotransformation, higher yields are reached. 

Dihydropyrimidinases (EC 3.5.2.2) are involved in the reductive pathway of pyrimidine degradation, catalyzing the hydrolysis of 5,6-DHU and 5,6-dihydrothymine to the corresponding Namino adds. However, dihydropyrimidinases have been more commonly known as hydantoinases [32, 33], as this enzyme can be used in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins using the so-called hydantoinase process, ... 

Although at present this chemoenzymatic method might not seem as versatile as the original hydantoinase process, the results on dihydropyrimidinases indicate a clear opportunity for the production of enantioenriched or enantiopure and P -amino acids by kinetic resolution ivhen the enantioselectivity of the enzyme is very high. Thus, further research is needed to find (or create) new enzymes with higher enantioselectivity. 

Clemente-Jimenez, J.M., Martinez-Rodriguez, S., Rodriguez-Vico, F., and Heras-Vazquez, F.J. (2008) Optically pure alpha-amino acids production by the Hydantoinase Process . Recent Patents Biotechnol., 2 (1), 35 46. 

C.-H. Kao, H.-H. Lo, S.-K. Hsu, W.-H. Hsu, A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine, I. Biotechnol. 134 (2008) 231-239. 

Hydantoinase process, outlined in Fig. 1, includes two hydrolases—hydantoin-hydrolyzing enzyme (hydantoinase) and AT-carbamoyl amino acid-hydrolyzing enzyme (carbamoylase)—and is one of the most efficient and versatile methods for the production of optically active a-amino acids. DL-5-Monosubstituted hydantoins, which are used as common precursors for the chemical synthesis of DL-a-amino acids [1], are the starting material of this enzymatic process. Keto-enol tautomerism is a typical feature of the hydantoin structure. Under neutral conditions, the keto form is dominant in alkaline solution, enolization between the 4 and 5 positions can occur, as has been concluded from the fact that optically pure hydantoins readily racemize. This feature is of practical relevance for the complete conversion of racemic hydantoin derivatives to optically pure L- or D-a-amino acids without any chemical racemization step. A variety of hydantoinase and carbamoylase with different stereospecificity were found. They are D-specific hydantoinase (D-hydantoinase), L-specific hydantoinase (L-hydantoinase), none-specific hydantoinase (DL-hydantoinase), D-specific carbamoylase (D-carbamoylase), and L-specific carbamoylase (L-carbamoylase). With the combination of these enzymes, optically pure amino acids are obtained from DL-5-monosubstituted hydantoins (Fig. 2). The wide substrate range of hydantoinases and carbamoylases also gives generality to the hydantoinase process. 

Figure 2 Enzymatic reactions involved in hydantoinase process.

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