Immobilized enzyme amino acid acylase

The chemical synthesis of amino acids leads to a racemic mixture. Only the L-form is usable for medicines and foodstuffs. Immobilized enzymes have been used for the industrial separation of enantiomers of various amino acids, e.g., amino acid acylase immobilized onto DEAE-Sephadex. Processing efficiency in the fermentation of amino acids has been greatly facilitated through the use of immobilized amino acid acylases such as L-glutamate aminoacylase. 

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. 

Acylase (acylase I aminoacylase N-acetyl amino acid amidohydrolase E.C. 3.5.1.14), is one of the best-known enzymes as far as substrate specificity (Chenault, 1989) or use in immobilized (Takahashi, 1989) or membrane reactors (Wandrey, 1977, 1979 Leuchtenberger, 1984 Bommarius, 1992a) is concerned however, its exact mechanism or 3D structure is still not known (Gentzen, 1979 1980). Acylase is available in large, process-scale quantities from two sources, porcine kidney and the mold Aspergillus oryzae. 

A recent example of a racemate separation process (D,L-methionine) by immobilized acylase has been described 53) indicating the upcoming trend of enzyme technology in amino acid production (Fig. 2, Table 3). 

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. 

Penicillins and cephalosporins are charaeterized by (3-lactam structures and are the antibiotics that have traditionally been those most eommonly used in the treatment of infeetions. Pharmaceutical companies have synthesized a variety of semisynthetic (3-lactam compounds for use as oral antibiotics, for example, ampicillin and amoxieillin. These penicillin derivatives are prepared by aeylation of 6-amino-penicillanie acid (6-APA) derived from penicillin G (benzyl penicillin) or penicillin V (phenoxymethyl penicillin). An immobilized penicillin amidase (penicillin acylase) from Escherichia coli or Bacillus megaterium is used to prepare the 6-APA in nearly quantitative yield (Fig. 4). This substance is used as the starting material for the produetion of a number of other penieillins. The immobilized enzyme can be reused more than... 

In the same group as nitrilase enzymes are the amidases. This includes amino acid amidase (EC 3.5.1.4), used to prepare amino acids, usually through resolution, and also penicillin G acylase (penicillin G amidohydrolase) (EC 3.5.1.11), used in the manufacture of semisynthetic penicillins [102,103]. Immobilized penicillin G acylase has most recently been used to catalyze the formation of A-a-phenylacetyl amino acids, which can then be used in peptide coupling reactions (see Section 13.2.3.2) [104]. 

Fig. 6. Kinetics of immobilization of glutaryl-7-ACA-acylase on epoxy-activated polymethacrylate. The Gl-7-ACA-acylase was incubated with the epoxy-activated carrier. At definite times aliquots were taken from the reaction suspension. Supernatant and carrier-fixed enzyme were separated by centrifugation. The carrier-fixed enzyme was washed with water to remove non-covalently linked enzyme. The activities of the immobilized enzyme and supernatant were determined (5 mM potassium phosphate buffer pH 8,37°C, 2% glutaryl-7-amino cepha-losporanic acid, pH-stat 8.0). Simultaneously, an aliquot of carrier-fixed enzyme was boiled in sodium dodecylsulfate (SDS)/glycine buffer and the supernatant was subjected to SDS-polyacrylamide electrophoresis (see insert from left to right lane 1 Carrier-fixed enzyme, 2 h lane 2 Carrier-fixed enzyme, 4 h lane 3 Carrier-fixed enzyme, 6 h lane 4 Carrier-fixed enzyme, 21 h lane 5 Carrier-fixed enzyme, 69 h lane 6 Dialyzed enzyme lane 7 Supernatant, 2 h lane 8 Supernatant, 21 h lane 9 Supernatant, 69 h lane 10 Molecular weight calibration markers)...

It is also noted that Chibata and his colleagues [14] of Tanabe Pharmaceutical Co. started to use an immobilized enzyme for the optical resolution of DL-amino acids in 1969. The process included a fungal acylase immobilized on DEAE-Sephadex to hydrolyze N-acyl-L-amino acids selectively. This was the first industrial use of immobilized enzymes leading to the present concept of bioreactors. 

Recently, a similar process has been applied by Degussa for the production of L-amino acids. In this case, L-amino acids are obtained by biocatalytic division of synthetically-produced acetyl DL-amino acids by means of enzymes. Unlike the previous type of fixed-bed reactor with carrier-located acylase, the new approach employs the enzyme in soluble form, and uses a membrane for separating the enzyme from the reagent solution. This avoids losses at the immobilizing stage and reduces enzyme consumption (.5). 

Biocatalytic resolution plays a major role in the industrial scale synthesis of a wide variety of optically pure amino acids. Tanabe uses an L-spe-cific aminoacylase for the manufacture of several L-amino acids, immobilized on DEAE-Sephadex. Degussa on the other hand, uses the free acylase in a membrane bioreactor. The process is highly efficient in enzyme use, and racemisation of the D-isomer is straightforward, thus providing good economics, and virtually no waste (Scheme 7.4). The process can be further refined by the use of racemase enzymes, which makes dynamic kinetic resolution feasible. 

The first commercial use of immobilized enzymes was developed in the mid-1960s, when amino acylase was used to separate racemates of L-amino acids by adsorption. Next came penicillin amadase for the production of semisynthetic penicillins. A major industrial application of immobilized enzymes was achieved in 1976 with the use of glucose isomerase to convert glucose to fructose. The first product was Sweetzyme developed by Novo Nordisk. Glucose was available as a by-product from the manufacture of cornstarch, but was not used as a sweetener because of its relative lack of sweetness (93). Because fructose tastes more than... 

Penicillin acylase is an extremely important enzyme for the industrial production of 6-aminopenicillanic acid and 7-amino 3-desacetoxicephalosporanic, as key intermediates of semi-synthetic (3-lactam antibiotics (Parmar et al. 2000). These precursors are now industrially produced mainly by hydrolysis of penicillin G and cephalosporin G with immobilized penicillin acylase, which have replaced the former cumbersome chemical processes almost completely (Bruggink 2001 Kallenberg et al. 2005), representing one of the most successful cases of industrial application of hydrolytic enzymes in bioprocesses. 

Ospina S, Lopez-Munguia A, Gonzalez R et al. (1992) Characterization and use of a penicillin acylase biocatalyst. J Chem Technol Biotechnol 53 205-214 Ospina S, Barzana E, Ramirez O et al. (1996) Effect of pH in the synthesis of ampicilfinby penicillin acylase. Enzyme Microb Technol 19 462-469 Palazzi E, Converti A (2001) Evaluation of diffusional resistances in the process of glucose isomerization to fructose by immobilized glucose isomerase. Enzyme Microb Technol 28 246-252 Pan JL, Syu MJ (2004) A thermal study on the use of immobilized penidllin G acylase in the formation of 7-amino-3-deacetoxy cephalosporanic acid from cephalosporin G. J Chem Technol Biotechnol 79(10) 1050-1056... 

Batchelor FR, Doyle FP, Nayler JHC et al. (1959) Synthesis of penicillin 6-amino penicUlanic acid in penicillin fermentations. Nature 183 257-258 Bmggink A (2001) Synthesis of 3-lactam antibiotics. Kluwer Acad Publ, Dordrecht, 335 pp Bmggink A, Roos EC, de Vroom E (1998) Penicillin acylase in the industrial production of P-lactam antibiotics. Org Proc Res Develop 2 128-133 Bryjak J, Trochimczuk AW (2006) Immobilization of lipase and penicillin acylase on hydrophobic carriers. Enzyme Microb Technol 39 573-578... 

An enzyme-labile so-called safety catch linker 452 was used successfully in various palladium-catalyzed cross-coupling reactions [592]. The linker 452, which releases a hydroxy or an amino functionality on enzymatic cleavage of its phenylacetamide moiety and subsequent rapid lactam formation, was attached to a soluble POE 6000 (polyethylene oxide) polymer and its free phenylacetic acid moiety was transformed to an m-iodobenzyl ester. The thus immobilized m-iodobenzyl alcohol was Heck-coupled with tert-butyl acrylate, and the coupling product 453 was cleaved off the solid support with penicillin G acylase under very mild conditions (pH 7, 37°C) (Scheme 8.84). 

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