The Amidase Process

While the production of D-amino acids is well established the preparation of L-amino acids is difficult due to the limited selectivity and narrow substrate spectrum of L-hydantoinases. This can be circumvented by employing rather un-selective hydantoinases in combination with very enantioselective L-carbamoyl-ases and carbamoyl racemases [90]. Furthermore, a D-hydantoinase has been genetically modified and converted into a L-hydantoinase. This enzyme can be used on a 100-kg scale for the production of L-tert-leucine [34]. Finally, the fact that the X-ray structure of an L-hydantoinase is known gives hope that side-directed mutagenesis will lead to improved L-hydantoinases [91]. 

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- 

provided all necessary biocatalysts are or were available, either d- or i-amino acids can be synthesized. Straightforward synthesis of most racemic 5-monosub-stituted hydantoins from inexpensive and often readily available starting materials through the following reactions provides another advantage for the process (Syldatk, 1999)  

As evidenced by the well-established industrial production of D-amino acids (mostly D-phenylglycine and p-OH-D-phenylglycine by the hydantoinase/carbamoylase route), both D-hydantoinases and D-carbamoylases are well developed. Enantio-selectivity in most cases is not a problem (Gross, 1987). 

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)  

4 Reductive Amination of Keto Acids (L-tert-Leudne as Example) 

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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. 

There are two distinct classes of enzymes that hydrolyze nitriles. Nittilases (EC 3.5.5. /) hydrolyze nittiles directiy to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nittiles also may be first hydrated by nittile hydratases to yield amides which are then converted to carboxyUc acid with amidases. This is a two-enzyme process, in which enantioselectivity is generally exhibited by the amidase, rather than the hydratase. 

To accelerate the polymerization process, some water-soluble salts of heavy metals (Fe, Co, Ni, Pb) are added to the reaction system (0.01-1% with respect to the monomer mass). These additions facilitate the reaction heat removal and allow the reaction to be carried out at lower temperatures. To reduce the coagulate formation and deposits of polymers on the reactor walls, the additions of water-soluble salts (borates, phosphates, and silicates of alkali metals) are introduced into the reaction mixture. The residual monomer content in the emulsion can be decreased by hydrogenizing the double bond in the presence of catalysts (Raney Ni, and salts of Ru, Co, Fe, Pd, Pt, Ir, Ro, and Co on alumina). The same purpose can be achieved by adding amidase to the emulsion. 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

This may be illustrated by the following process, catalyzed by penicillin amidases (EC 3.5.1.1 1) from various sources... 

The optimum yield of a condensation product is obtained at the pH where Ka has a maximum. For peptide synthesis with serine proteases this coincides with the pH where the enzyme kinetic properties have their maxima. For the synthesis of penicillins with penicillin amidase, or esters with serine proteases or esterases, the pH of maximum product yield is much lower than the pH optimum of the enzymes. For penicillin amidase the pH stability is also markedly reduced at pH 4-5. Thus, in these cases, thermodynamically controlled processes for the synthesis of the condensation products are not favorable. When these enzymes are used as catalysts in thermodynamically controlled hydrolysis reactions an increase in pH increases the product yield. Penicilhn hydrolysis is generally carried out at pH about 8.0, where the enzyme has its optimum. At this pH the equiUbrium yield of hydrolysis product is about 97%. It could be further increased by increasing the pH. Due to the limited stability of the enzyme and the product 6-aminopenicillanic acid at pH>8, a higher pH is not used in the biotechnological process. 

Immobilized forms of penicillin amidases and acylases have replaced whole-cell biocatalysts for the production of 6-APA and 7-ACA as they can be reused many times, in some cases for over 1000 cycles. Another major advantage is the purity of the enzyme, lacking the /3-lactamase contaminants often present in whole cells. The productivity of these biocatalysts exceeds 2000 kg prod-uct/kg catalyst. A typical process for the production of 6-APA employs immobilized penicillin G acylase covalently attached to a macroporous resin. The process can be run in either batch or continuous modes. The pH of the reaction must be maintained at a value between 7.5 and 8 and requires continuous adjustment to compensate for the drop caused by the phenylacetic acid generated during the course of the reaction. Recycle reactors have been used, as they allow both pH control and the use of packed bed reactors containing the immobilized catalyst. The enzymatic process is cheaper, although not... 

The starting material for the acylase process is a racemic mixture of N-acetyl-amino acids 20 which are chemically synthesized by acetylation of D, L-amino acids with acetyl chloride or acetic anhydride in alkaU via the Schotten-Baumann reaction. The kinetic resolution of N-acetyl-D, L-amino acids is achieved by a specific L-acylase from Aspergillus oryzae, which only hydrolyzes the L-enantiomer and produces a mixture of the corresponding L-amino acid, acetate, and N-acetyl-D-amino acid. After separation of the L-amino acid by a crystallization step, the remaining N-acetyl-D-amino acid is recycled by thermal racemization under drastic conditions (Scheme 13.18) [47]. In a similar process racemic amino acid amides are resolved with an L-spedfic amidase and the remaining enantiomer is racemized separately. Although the final yields of the L-form are beyond 50% of the starting material in these multistep processes, the effidency of the whole transformation is much lower than a DKR process with in situ racemization. On the other hand, the structural requirements for the free carboxylate do not allow the identification of derivatives racemizable in situ therefore, the racemization requires... 

This was performed for each enzyme independently, feeding the reactor with the appropriate substrate (nitrile for the cascade reaction, amide for the sole amidase). The activation energies of both catalysed reactions were evaluated together with those of the inactivation process that inevitably takes place even under the most suitable operational conditions. In the nitrile hydratase/amidase cascade system nitrile hydratase is the more labile enzyme that imposes process temperature choice. These findings make accessible the complete kinetic expression of the dependence from temperature of reaction rate, allowing accurate prediction on reactor performances for process scale-up. 

Production of Amino Acids Using Amidases the DSM Process... 

Several multi-ton industrial processes still use enzymatic resolution, often with lipases that tolerate different substrates. BASF, for example, makes a range of chiral amines by acylating racemic amines with proprietary esters. Only one enantiomer is acylated to an amide, which can be readily separated from the unreacted amine. Many fine chemicals producers also employ acylases and amidases to resolve chiral amino acids on a large scale. l-Acylases, for example, can resolve acyl d,l-amino acids by producing the I-amino acids and leaving the N-acyl-l-amino acid untouched after separation, the latter can be racemized and returned to the reaction. d-Acylase forms the alternative product. Likewise, DSM and others have an amidase process that works on the same principle d,l-amino acid amides are selectively hydrolyzed, and the remaining d-amino acid amide can be either racemized or chemically hydrolyzed. 

This resulted in a two-step, one pot biotransformation. In this process the gene for the amidase was also cloned into Escherichia coli, resulting in a number of additional advantages ... 

In the first step the (R,S)-nitrile is rapidly and quantitatively hydrolyzed to the (R,S)-amide. The amidase containing biomass is then added so that the (R)-amide is specifically hydrolyzed to the (R)-acid, and the product, the (S)-amide, remains. A completely new isolation process was developed ultra-filtration, electrodialysis, ion-exchange chromatography, reverse osmosis, crystallization, centrifugation, and drying. The product has been produced at a 15 m3 scale and has an ee value of >98%. The isolated yield calculated from the nitrile was >35%. The (R)-acid can be recycled by reacting it with thionyl chloride and ammonia to produce the (R,S)-amide. This results in minimal waste and higher yields. 

The enzymatic process uses water as the solvent and two immobilized enzymes as catalysts at room temperature. In a first step cephalosporin C is deaminated to a-ketoadipyl-7-ACA using a carrier-fixed D-amino acid oxidase in the presence of oxygen. Under reaction conditions the a-keto intermediate is oxidatively decarboxy-lated to glutaryl-7-ACA. In a second step the glutaryl-7-ACA is then hydrolyzed to 7-ACA by a carrier-fixed glutaryl amidase. 

It should also be noted that at DSM this amidase process has been extended towards the synthesis of optically active a,a-disubstituted amino acids. For example, the antihypertensive drug L-methyl-dopa, l-10, has been produced successfully (Fig. 8) [14, 15]. 

Fig. 7 The DSM amidase process for enantiomerically pure a-amino acids.

Many pesticides are esters or amides that can be activated or inactivated by hydrolysis. The enzymes that catalyze the hydrolysis of pesticides that are esters or amides are esterases and amidases. These enzymes have the amino acid serine or cysteine in the active site. The catalytic process involves a transient acylation of the OH or SH group in serin or cystein. The organo-phosphorus and carbamate insecticides acylate OH groups irreversibly and thus inhibit a number of hydrolases, although many phosphorylated or carbamoylated esterases are deacylated very quickly, and so serve as hydrolytic enzymes for these compounds. An enzyme called arylesterase splits paraoxon into 4-nitrophenol and diethyl-phosphate. This enzyme has cysteine in the active site and is inhibited by mercury(ll) salts. Arylesterase is present in human plasma and is important to reduce the toxicity of paraoxon that nevertheless is very toxic. A paraoxon-splitting enzyme is also abundant in earthworms and probably contributes to paraoxon s low earthworm toxicity. Malathion has low mammalian toxicity because a carboxyl esterase that can use malathion as a substrate is abundant in the mammalian liver. It is not present in insects, and this is the reason for the favorable selectivity index of this pesticide. 

Later in the process this side chain is removed enzymatically, using an enzyme quite similar to the glutaryl amidase from Pseudomonas sp. as in the enzymatic production of 7-ACA. For the production of 7-ADCA and the dicarboxylic acid amidase, new plants are currently under construction at DSM in The Netherlands. Compared with the old process for the production of 7-ADCA, the major advantages of this process are higher purity of the end product, much greater energy efficiency and almost complete absence of organic solvents. 

The limited effect which we have just found with butyro-amidase acting on the products of hydrolysis of the nitrogenous materials, can be explained by the hypothesis that the bacterium secretes numerous specific enzymes, each acting on different substances. Butyro-amidase, thus isolated, would contain only certain enzymes, and not all those which are necessary for the evidently more exacting fermentation. Althou the amidases attack a series of substances of similar structure, they set ammonia free by different methods. We are, therefore, quite naturally led to believe that in this process several catalysts intervene, for it would be inadmissible that the same enzyme should be called upon to fill such varied functions. We will then conclude by saying that to each of the different chemical processes there corresponds a well-determined type of catalyst, whether the reaction be one of hydroly, or one of reduction. Unfortunately, in the present state of the question, this opinion cannot be confirmed by any experimental proof. 

In the experiments with yeast, it is the amidases in reserve inside the cells which act. Necessarily, we are led to use large quantities in order to obtain results. To obviate this inconvenience, which would render the process not very practical, bacteria are utilized that are capable of secreting amidases in abundance. The bacterial q>ecies chosen must be well determined, for not all the ammonia ferments lend themselves to the exacting work required by industry. Moreover, it is for many reasons indi nsable that the bacterium adopted shall be acclimated to the conditions of the environment so that the formation and the secretion of its catalysts shall be favored as much as possible. In fact, bacterial activity producing ammonia is alwa) very slow. The bacteria are very sensitive to their own products, and the deamidization which they cause is never... 

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