Proteases and Acylases

Considerable attention has also been given to enantioselective enzymatic hydrolysis of esters of a-amino acids. This is of particular importance as a means of preparing enantiopure samples of unusual (non-proteinaceous) a-amino acids. The readily available proteases a-chymotrypsin (from bovine pancreas) and subtilisin (from Bacillus lichenformis) selectively hydrolyze the L-esters, leaving D-esters unreacted. These enzymatic hydrolysis reactions can be applied to V-protected amino acid esters, such as those containing r-Boc and Cbz protecting groups. 

Much of the interest in acylases originated from work with the penicillins. Structurally modified penicillins can by obtained by acylation of 6-aminopenicillamic acid. For example, the semisynthetic penicillins such as amoxicillin and ampicillin are obtained using enzymatic acylation. Acylases are used both to remove the phenyl-acetyl group from the major natural penicillin, penicillin G, and to introduce the modified acyl substituent. 

Barrett, N. D. Rawlings, and J. F. Woessner, eds.. Handbook of Proteolytic Enzymes, 2nd Edition, Elsevier, 2004. 

Polgar in Mechanisms of Protease Action, CRC Press, Boca Raton, FL, Chap. 3, 1989 J. J. Perona and C. S. Craik, Protein ScL, 4 337 (1995). 

Schricker, K.Thirring, and H. Berner, Biorg. Med. Chem. Lett., 2, 387 (1992). 

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The seminal work of Klibanov in the early 1980s [46,47] made it clear that enzymes can be used in hydrophobic organic solvents, although at the price of a severely reduced reaction rate [48, 49]. Indeed, many Upases, as well as some proteases and acylases, are so stable that they maintain their activity even in anhydrous organic solvents. This forms the basis for their successful application in non-hydrolytic reactions, such as the (enantioselective) acylation of alcohols and amines, which now are major industrial applications [50]. 

A particularly elegant solution for the assay of proteases and acylases is offered by the fluorogenic detection of free amino acids by decomplexation of copper from calcein, which removes its quenching effect This principle has been used for assays of acylases, amidases, and proteases (Scheme 1.13) [52, 53]. For the case of proteases combinatorial assays are particularly in demand for testing multiple peptides in parallel and determining the cleavage specificity [54]. New solutions... 

In Japan, solid fermentation is still used to produce many kinds of enzymes including lipases, proteases and acylases. Some glycotransferases are also produced by solid fermentation. In the production of proteases, solid fermentation is often used to increase the productivity in solids. On changing to liquid fermentation, the protease is not produced and its properties change. Solid fermentation is old-fashioned and difficult to scale up because of the expensive facilities needed. 

The great chemical stability and lack of reactivity under mild conditions for deprotection has greatly hampered the widespread application of amides in protecting group chemistry. However, recent developments in this area may suggest that proteases and amidohydrolases of broad substrate specificity can serve to overcome these drawbacks. Thus, 3-phenylpropionamides and iV-benzoylphenylalanine amides are attacked by chymotrypsin (pH 7, 37 °C). The application of penicillin acylase for the selective removal of the phenylacetamido residue from peptides (23 equation 7) and carbohydrates is also promising. 

In biocatalysis, hydrolases are the most important class of enzymes for carrying out enzymatic resolutions. Many hydrolases, such as esterases, lipases, epoxide hydrolases, proteases, peptidases, acylases, and amidases, are commercially available a substantial number of them are bulk enzymes [87]. Resting-cell systems, if they are not immobilized, are used in diluted suspensions and could be handled as quasi-homogeneous catalysts. 

These catalyze the hydrolytic cleavage of bonds. Many commercially important enzymes belong to this class, e.g. proteases, amylases, acylases, lipases, and esterases. 

In addition to hydrol5dic enzymes such as lipases, proteases, and esterases, now more enz3rmes, such as I EDs, amino acid dehydrogenases, transaminases, nitrilases, acylases, amino acid dehydrogenases, and amidases are available. 

Traditional techniques such as physical adsorption and covalent linkage onto solid supports, entrapment in polymer matrices, and microencapsulation have long been used for immobilizing such enzymes as lipases, proteases, hydantoinases, acylases, amidases, oxidases, isomerases, lyases, and transferases [12-18]. Encapsulation and adsorption have also proved their utility in the immobilization of bacterial, fungal, animal, and plant cells [12-21]. However, as biocatalysis applications have grown, so the drawbacks and limitations of traditional approaches have become increasingly evident. The forefront issues now facing bioimmobilization are indicated in Table 1. 

Historically, the most popular enzymes used for chemical synthesis are lipases, esterases, proteases, acylases and amidases, among others. Recently, a number of recombinant biocatalysts have been discovered and isolated, significantly expanding the toolbox for biotransformations. In this section, the focus will be on these new enzymes. 

The use of enzymes and whole cells as catalysts in organic chemistry is described. Emphasis is put on the chemical reactions and the importance of providing enantiopure synthons. In particular kinetics of resolution is in focus. Among the topics covered are enzyme classification, structure and mechanism of action of enzymes. Examples are given on the use of hydrolytic enzymes such as esterases, proteases, lipases, epoxide hydrolases, acylases and amidases both in aqueous and low-water media. Reductions and oxidations are treated both using whole cells and pure enzymes. Moreover, use of enzymes in sngar chemistiy and to prodnce amino acids and peptides are discnssed. 

Industrial enzymes, such as amylases, proteases, glucose isomerase, lipase, catalases, and penicillin acylases... 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

Enzymes such as proteases (122), subtilisin (123), acylases, peptidases, amidases, and lipases (124) are reported to catalyze amide bond formation with, in some cases, enantiospeciflcity of over 99%. Despite limited enzyme-substrate compatibility, specific conditions have been developed to reverse their natural reactivity, which is in favor of the hydrolysis. For example, Kyotorphin (Tyr-Arg) (125), a potent analgesic, was produced on an industrial scale using a-chymotrypsin, a peptidase isolated from bovine pancreas. 

For the N-terminal deprotection of peptides, the enzyme penicillin G acylase from E. coli has been applied. This attacks phenylacetic acid (PhAc) amides and esters but does not hydrolyze peptide bonds [12-14,25]. The danger of a competitive cleavage of the peptide backbone at an undesired site, which always exists when proteases like trypsin and chymotrypsin are used, is overcome by using the acylase. The penicillin G acylase accepts a broad range of protected dipeptides (27) as substrates, and selectively liberates the N-terminal amino group under almost neutral conditions (pH 7-8, room temperature), leaving the peptide bonds as well as the C-terminal methyl-, allyl-, benzyl-, and tert-butyl ester unaffected (Fig. 8) [25a,bj. On the other hand, the phenylacetamide... 

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