Carbamoylase process

In the fine chemicals industry, enantiomerically pure amino acids are mainly produced by the aminoacylase process, the amidase process, and the hydantoinase/ carbamoylase process, all three of which are suitable for I- and D-amino acids. Dehydrogenases and transaminases are now becoming established for reduction processes. 

Fig. 31.17. Hydantoinase/Carbamoylase process for the production of D-amino acids.

The use of the whole cell system is necessary due to the limited stability of the carbamoyl-hydrolyzing enzyme and to the different requirements for optimal reactivity. Best carbamoylase activity is obtained by lowering the reaction pH slightly, which is regulated from the development of carbon dioxide from the first reaction step. The D-hydantoinase-D-carbamoylase process has proved successful for the preparation of a large number of D-amino acids [37] (Scheme 13.13). 

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. 

In contrast to the D-branch, application of i-hydantoinases and i-carbamoylases has not gone beyond small pilot scale yet. The enantioselectivity of most i-hydan-toinases varies depending on the substitution in the 5-position and can even cross over to the D-side (Nishida, 1987). In addition, i-carbamoylases are often very unstable (Cotoras, 1984). Over several years, the following steps were taken to improve the process to economically necessary levels (May, 2002) ... 

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]. 

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. 

In addition, the use of a whole-cell biocatalyst consisting of a racemase, hydantoi-nase, and carbamoylase allows a dynamic biocatalytic resolution. Besides resolution processes, asymmetric (bio-)catalytic concepts have been applied successfully on an industrial scale. The different types of asymmetric (bio-)catalytic syntheses of L-amino acids, based on the use of prochiral starting materials, are shown in Fig. 3. 

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. 

In summary, a broad range of large-scale applicable biocatalytic methodologies have been developed for the production of L-amino acids in technical quantities. Among these industrially feasible routes, enzymatic resolutions play an important role. In particular, L-aminoacylases, L-amidases, L-hydantoinases in combination with L-carbamoylases, and /l-lactam hydrolases are efficient and technically suitable biocatalysts. In addition, attractive manufacturing processes for L-amino acids by means of asymmetric (bio-)catalytic routes has been realized. Successful examples are reductive amination, transamination, and addition of ammonia to rx,/fun-saturated carbonyl compounds, respectively. 

The enzymatic process of d-HPG production was started in 1980 in Singapore, and the immobilized D-carbamoylase reactor was introduced in 1995. The amount of production of d-HPG by this method is around 2000 tons/year [26]. 

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. 

Kaneka Co. Ltd. started the enzymatic production of D-p-hydroxyphenylglycine (d-HPG) in 1980 in Singapore, and the immobilized D-carbamoylase reactor was introduced in this process in 1995. The annual production of d-HPG by this method is around 2,000 t. d-HPG is a starting material for the production of semisynthetic penidllins and cephalosporins, such as amoxicillin and cefadroxil. d-HPG is produced from DL-p-hydroxyphenylhydantoin (DL-HPH) by a two-step enzymatic method (Takahashi 1986). 

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. 

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