Soluble acylase

Figure 12.3-3. Stability of soluble acylase I in the presence of organic cosolvents. Acylase I from porcine kidney (a, c) and from Aspergillus (b, d) were incubated in 0,10 m phosphate buffer, pH 7.5, containing ethanol (a, b) or DM SO (c, d), at 25 °C, under nitrogen. Concentrations of organic cosolvents were 10% ( ), 20% (o), 30% ( ), 40% ( ), and 50% (a)1 601.

An alternative to extraction crystallization is used to obtain a desired enantiomer after asymmetric hydrolysis by Evonik Industries. In such a way, L-amino acids for infusion solutions or as intermediates for pharmaceuticals are prepared [35,36]. For example, non-proteinogenic amino acids like L-norvaline or L-norleucine are possible products. The racemic A-acteyl-amino acid is converted by acylase 1 from Aspergillus oryzae to yield the enantiopure L-amino acid, acetic acid and the unconverted substrate (Figure 4.7). The product recovery is achieved by crystallization, benefiting from the low solubility of the product. The product mixture is filtrated by an ultrafiltration membrane and the unconverted acetyl-amino acid is reracemized in a subsequent step. The product yield is 80% and the enantiomeric excess 99.5%. 

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). 

All the transformations carried out with penicillin acylase and employing phenylacetates or -amides as substrates are hampered by the very limited solubility of these esters in aqueous environments. Although the enzyme tolerates considerable amounts of organic cosolvents, generally their application results in at least a partial deactivation of the enzyme. Since penicillin acylase accepts variations in the phenylacetic acid part of its substrates, pyridyl acetic acid esters were employed to enhance the solubihty of the substrates in aqueous solution. In fact, several simple 4-pyridylacetates turned out to be fairly soluble in aqueous media and were attacked at very acceptable rates by the enzyme [ 19 ]. It is interesting to note that the velocity of the enzymatic transformations depends... 

Resolution of DL-alanine (1) is accomplished by heating the N-acetyl derivative (2) in weakly alkaline solution with acylase, a proteinoid preparation from porcine kidney containing an enzyme that promotes rapid hydrolysis of N-acyl derivatives of natural L-amino acids but acts only immeasurably slowly on the unnatural o-isomers. N-Acetyl-DL-alanine (2) can thus be converted into a mixture of l-( + )-alanine (3) and N-acetyl-o-alanine (4). The mixture is easily separable into the components, because the free amino acid (3) is insoluble in ethanol and the N-acetyl derivative (4) is readily soluble in this solvent. Note that, in contrast to the weakly levorotatory o-( - )-alanine (a 14.4°), its acetyl derivative is strongly dextrorotatory. 

In case the yield of L-alanine is low, evaporation of this mother liquor may reveal the reason. If the residue solidifies readily and crystallizes from acetone to give acetyl-DL-alanine, mp 130°C or higher, the acylase preparation is recognized as inadequate in activity or amount. Acetyl-D-alanine is much more soluble and slow to crystallize. 

Aminoacylase has also been immobilized on a nylon membrane1661. While the half-life as measured by thermal stability, of 161 d is superior to the data for immobilized acylase (65 d)[6] or soluble enzyme in an EMR191, reactor productivity at 0.136 L-valine kg/L-1d-1 is lower than that for DEAE-Sephadex-immobilized acylase (0.5 kg/L-1d-1)161 or that for a membrane reactor (0.35 kg/L-1d j191. 

The N-acetyl-D,L-amino acid precursors are conveniently accessible through either acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in a Schotten-Baumann reaction or via amidocarbonylation I801. For the acylase reaction, Co2+ as metal effector is added to yield an increased operational stability of the enzyme. The unconverted acetyl-D-methionine is racemized by acetic anhydride in alkali, and the racemic acetyl-D,L-methionine is reused. The racemization can also be carried out in a molten bath or by an acetyl amino acid racemase. Product recovery of L-methionine is achieved by crystallization, because L-methionine is much less soluble than the acetyl substrate. The production is carried out in a continuously operated stirred tank reactor. A polyamide ultrafiltration membrane with a cutoff of 10 kDa retains the enzyme, thus decoupling the residence times of catalyst and reactants. L-methionine is produced with an ee > 99.5 % and a yield of 80% with a capacity of > 3001 a-1. At Degussa, several proteinogenic and non-proteinogenic amino acids are produced in the same way e.g. L-alanine, L-phenylalanine, a-amino butyric acid, L-valine, l-norvaline and L-homophenylalanine. 

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). 

Adsorption is a simple and straightforward route for biocatalyst immobilization which offers unique advantages over soluble enzymes, such as enhanced activity, increased selectivity, improved stability and reusability. For example, Assemblase, the commercial name of immobilized pencillin-G acylase from E. coli, has been used by industry for manufacture of the semi-synthetic p-lactam antibiotic cephalexin. ... 

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). 

Biocatalysts that are reversibly soluble as a function of pH have been obtained by the covalent coupling of lysozyme to alginate (113) of trypsin to poly(acrolein-co-acrylic acid) (114) and of cellulase (115), amylase (115) a-chymotrypsin, and papain (116) to poly(methyl methacrylate-co-methacrylic acid). A reversibly soluble cofactor has been produced by the covalent binding of NAD to alginate (117). Reversibly soluble a-chymotrypsin, penicillin acylase, and alcohol dehydrogenase were produced by coupling to the polycation component of polyelectrolyte complexes formed by poly(methacrylic acid) and poly(iY-ethyl-4-vinyl-pyridinium bromide) (118). 

Amino acylases catalyse the hydrolysis of N-acyl-L-aminoacids (Figure 6.28) but not of the corresponding D-isomers. This reaction is comparable to that catalysed by penicillin acylase which in fact shows a similar selectivity when hydrolysing amides other than penicillin G. If the racemic mixture of an amino acid is first acylated and is then treated with the acylase, the product will be a mixture of optically pure L-amino acid and predominantly D-acyl amino acid. These are easily separated on the basis of their solubilities at the isoelectric point of the amino acid. The pure L-amino acid can be crystallized from the mixture leaving the residual acyl amino acid to be racemized and recycled through the process. 

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