The Acylase Process

A straightforward approach to avoid low yields is to perform the reaction as a dynamic kinetic resolution. Racemisation can be achieved chemically [33] or enzymatically, indeed a number of N-acyl amino acid racemases have been described and it has been demonstrated that they could be employed together with the l-N-acyl amino acylase for the production of optically pure methionine [81]. 

The acylase process can also be applied for the production of D-amino acids. These amino acids are valuable building blocks in pharmaceutical chemistry and they can be prepared with high enantiopurity by the action of a D-N-acyl 

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A. S. Bommarius, M. Schwarm and K. Drauz, The membrane reactor for the acylase process -from laboratory results to commercial scale, Chim. Oggi 1996, 14(10), 61-64. 

The starting material for the acylase process is a racemic mixture of N-acetyl amino acids which are chemically synthesized, most conveniently by acetylation of... 

The acylase-catalyzed resolution of N-acetyl-D,L-amino acids to obtain enantiomerically pure i-amino acids (see Chapter 7, Section 7.2.1) has been scaled up to the multi-hundred ton level. For the immobilized-enzyme reactor (Takeda, 1969) as well as the enzyme membrane reactor technology (Degussa, 1980) the acylase process was the first to be scaled up to industrial levels. Commercially acylase has broad substrate specificity and sufficient stability during both storage and operation. The process is fully developed and allowed major market penetration for its products, mainly pharmaceutical-grade L-methionine and L-valine. 

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... 

The acylase process is a typical example of reaction and separation by membranes in a sequential mode. The process is fully developed up to industrial scale, yielding high-quality products at good cost effectiveness. 

Enzymatic resolution is another method applied for the production of l- as well as D-amino acids. The disadvantage of a resolution process, a maximal yield of 50%, can often be overcome by racemization of the unwanted enantiomer of the amino acid derivative (vide infra). Many resolution concepts have been developed and commercialized, e.g., the acylase process by Degussa and Tanabe [15], the D-hydantoinase process by Recordati and Ajinomoto [16], the aminoamidase processes of DSM, and different lipase-catalyzed processes [1,13b, 17]. 

In this process, penicillin G is first hydrolysed to 6-APA with the acylase derived from Kluyvera citwphila at a slightly alkaline pH (pH 75). Subsequently the 6-APA is incubated with an acylase derived from Pseudomonas mdanogenum and with DL-phenylglydne methyl ester at pH 55. This produces ampiciilin in reasonable yields only because of the specificity of the P. melanogenum enzyme. This enzyme does not react with penicillin G nor phenylacetic acid. 

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

Degussa AG uses immobilised acylase to produce a variety of L-amino adds, for example L-methionine (80,000 tonnes per annum). The prindples of the process are the same as those of the Tanabe-process, described above. Degussa uses a new type of reactor, an enzyme membrane reactor, on a pilot plant scale to produce L-methionine, L-phenylalanine and L-valine in an amount of 200 tonnes per annum. 

Chemical reactions enhanced by catalysts or enzymes are an integral part of the manufacturing processes for the majority of chemical products. The total market for catalysts and enzymes amounts to 11.5 billion (2005), of which catalysts account for about 80%. It consists of four main applications environment (e.g., automotive catalysts), 31% polymers (e.g., polyethylene and polypropylene), 24% petroleum processing (e.g., cracking and reforming), 23% and chemicals, 22%. Within the latter, particularly the catalysts and enzymes for chiral synthesis are noteworthy. Within catalysts, BINAPs [i.e., derivatives of 2,2 -bis(diphenylphosphino) -1, l -bis-l,l -binaphthyl) have made a great foray into chiral synthesis. Within enzymes, apart from bread-and-butter products, like lipases, nitrilases, acylases, lactamases, and esterases, there are products tailored for specific processes. These specialty enzymes improve the volumetric productivity 100-fold and more. Fine-chemical companies, which have an important captive use of enzymes, are offering them to third parties. Two examples are described here ... 

The immobilized enzyme is more active and/or more stable than the soluble equivalent the immobilization process retards unfolding. Examples are glucose isomerase (GI) (Chapter 7, Section 7.3.1.) or penicillin G acylase (PGA) (Section 7.5.1). 

Figure 7.36 Schematic of the new process to semi-synthetic antibiotics with pen G acylase (Drauz, 2002).

Facing difficulties with chemical methods, development work was aimed at finding a biocata-lytic process to resolve the amino acids. Typical commercial enzymes reported for resolution of amino acids were tested. Whole-cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids 106 112 the acylase catalyzed resolution of Af-acyl amino acids had extremely low rates (often zero) of catalysis toward a-dialkylated amino acids 113114 and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-... 

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... 

Studies to scale up the fermentation process for FR901379 and FR901379 acylase . 

An example of an acylase to perform a resolution is provided by the Degussa process to L-methionine (1). The racemic acetylmethionine (2) is prepared by a chemical synthesis. The acylase hydrolyses only the L-isomer (Fig. 2). The D-isomer is racemized by base and put back into the process stream (48). 

Membrane technology is a well-established technology for the immobilization of enzymes [233] since Degussa [234] introduced a continuous acylase process employing an enzyme-membrane reactor for the enantiomeric production of pure L-amino acids in 1981. Polymer membranes configured into hollow-fiber modules are, by far, the most widely used membrane where the enzyme is held back by the low cutoff of the membrane. 

An example of a commercial process that uses a hydrolase in the hydrolytic mode is the production of the antibiotic intermediate 6-APA by Gist-brocades [13], discussed above in Section 7.2.1 (Fig. 7.1). The biocatalyst is a penicillin acylase, which generates 6-APA from the starting material penicillin G in one step at a moderate temperature in water. The atom utilization of the enzymic process is much higher than that of the corresponding chemical process [14]. 

In subsequent process generations, peniciUin G acylase derived enzymes were also used to couple the synthetic side chains, such as D-phenylglycine (ampicillin, cephalexin) and D-p-hydroxyphenylglycine (amoxicillin, cephadroxil) in the form of amino acid amides or esters to 6-APA and 7-ADCA (Scheme 4.6D). Biotransformation routes to the n-phenylglycine and n-p-hydroxyphenylglycine side chains were also developed (Scheme 4.6C), but the enzymatic process towards n-phenyl-glycine amide has been substituted by a classical resolution. 

An enzymatic procedure for amine resolution, employing acylation by C. antarctica lipase B and deacylation by penicillin G acylase, has been demonstrated by Ismail et al. (Figure 14.14) [20]. The acylase catalyzed deacylation provides a greener process than the standard chemical deacylation as a result of the elimination of the salt waste stream typically generated by deacylating under strongly alkaline condi tions. It is also more amenable to sensitive functional groups that are not stable under basic conditions. A drawback of this approach is that the amide hydrolysis step is... 

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