Penicillin G-acylase

This ester is conveniently formed from a penicillinic acid with PhCH2C02CH2Cl and TEA. Cleavage is accomplished by enzymatic hydrolysis with penicillin G. acylase in 70-90% yield. 

Penicillin G. acylase, pH 7.8 buffer, 35°, 30 min to 2 h. These conditions result in isolation of the disulfide, but if j8-mercaptoethanol is included in the reaction mixture, the thiol can be isolated. ... 

Preparation of PhAcOZ amino acids proceeds from the chloroformate, and cleavage is accomplished enzymatically with penicillin G acylase (pH 7 phosphate buffer, 25°, NaHS03, 40-88% yield). In a related approach, the 4-ace-toxy derivative is used, but in this case deprotection is achieved using the lipase, acetyl esterase, from oranges (pH 7, NaCl buffer, 45°, 57-70% yield). 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

Penicillin G acylase (PGA, EC 3.5.1.11, penicillin G amidase) catalyzes the hydrolysis of the phenylacetyl side chain of penicillin to give 6-aminopenicillanic acid. PGA accepts only phenylacetyl and structurally similar groups (phenoxyacetyl, 4-pyridylacetyl) in the acyl moiety of the substrates, whereas a wide range of structures are tolerated in the amine part [100]. A representative selection of amide substrates, which have been hydrolyzed in a highly selective fashion, is depicted in Figure 6.36. 

PSL, AMANO) has been found to catalyze the Markovnikov-type addition of thiols to vinyl esters (Scheme 5.23) [114] and acylases aminoacylase [115], penicillin G acylase [116], and acylase from AspergiUus oryzae [117], the same kind of addition of various nitrogen nucleophiles. Unfortunately, most additions described above are nonstereoselective. 

Patent literature reports on the analogous resolutions of phosphinotricin using, among others, penicillin G-acylase, penicillin G-amidase, subtilisin or microorganisms such as Enterobacter aerogenes, Klebsiella oxytoca, Corynebac-terium sp., Rhodococcus rubropertinctus and others7°... 

The above two processes employ isolated enzymes - penicillin G acylase and thermolysin, respectively - and the key to their success was an efficient production of the enzyme. In the past this was often an insurmountable obstacle to commercialization, but the advent of recombinant DNA technology has changed this situation dramatically. Using this workhorse of modern biotechnology most enzymes can be expressed in a suitable microbial host, which enables their efficient production. As with chemical catalysts another key to success often is the development of a suitable immobilization method, which allows for efficient recovery and recycling of the biocatalyst. 

Chong, A.S.M. and Zhao, X.S. (2004) Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst. Catalysis Today, 93-95, 293-299. 

Calcined [MgAl] LDH was also used to adsorb penicillin G acylase [121]. The calcined LDH phases have porous structures, large specific surface areas and abundant basic sites to bind the enzymes. The effect of varying the composition of the LDH precursor and calcination temperature on the activity of the immobilized enzyme has been reported. In this case, the percentage of immobilized proteins increases up to 88 %. 

A more sophisticated method was developed by Ren et al. [123] for the immobilization of penicillin G acylase by covalent grafting in the interlayer galleries of a LDH by a three-step procedure using a glutamate pillared LDH as the starting material. 

Penicillin acylases or amidohydrolases, which cleave the amide side chain of penicillin G, have been known for almost 50 years. " As one of the first enzymes to be developed for use at scale in the pharmaceutical industry, penicillin G acylase (PGA) has often been used as a model system for academic studies from molecular biology to biochemical engineering. Despite extensive screening, however, for decades there was no equivalent enzyme to generate 7-ACA by cleaving the polar D-a-aminoadipoyl side chain from cephalosporin C. 

Fernandez-Lafuente,R., RoseU, C.M., Piatkowska, B. and Guisan, J.M., Synthesis of antibiotics (cephaloglycin) catalyzed by penicillin G acylase evaluation and optimization of different synthetic approaches. Enzyme Microb. Technol., 1996, 19, 9-14. 

Pohl, T. and Waldmann, H., Enhancement of the enantioselectivity of penicillin G acylase from E. coli by substrate tuning . Tetrahedron Lett., 1995, 36, 2963-2966. 

Fig.1 Schematic illustration of the immobilization of penicillin G acylase on glutamate-pillared LDHs. Reaction of the primary amino groups of the intercalated glutamate ions with glutaraldehyde through Schiff s base formation is followed by covalent coupling of the residual aldehyde groups with the free amino groups of the enzyme...

Preparation of o-Amino Acids Using the W-Acylase Activity of Penicillin-G Acylase 85... 

In independent and simultaneous investigations Fhtsch et al. [41-42] and Wald-mann et al. [43—44] developed selectively cleavable exo-linkers, which can be cleaved with penicillin G acylase, a commercially available and widely used enzyme [45]. 

According to the safety-catch principle, the separation of the desired products proceeds in a two-step process. First, penicillin G acylase hydrolyses the phenyl-... 

The desired benzyl alcohols (67-69) were released by incubation of the corresponding polymer conjugates (64—66) with penicillin G acylase at pH 7 and 37 °C... 

Other immobilized enzyme product successes (where annual production of immobilized enzymes has surpassed 1 ton/year) comprise Aminoacylase (Amano), hydantoinase (Smith Kline Beecham), lactase (Valio), lipase (Novo Nordisk), penicillin G acylase (Gist-brocades, Smith Kline Beecham, Rohm) and penicillin V acylase (Novo Nordisk/Gist-brocades) (Poulsen, 1984). 

Up to 9 X10 ton/a is produced world-wide by a number of companies, snch as Beechams, Toyo Jozo, Pfizer, Gist-brocades (now DSM) etc. The majority is produced using penicillin G acylase, although some companies prefer to nse the V acylase. This large commercial potential was obviously a very big incentive to companies to develop this technology. 

As VAO is able to perform an oxidative deamination of capsaicin-derived vanillyl amine, vanillin can be produced by the pathway described in the previous subsection. Van den Heuvel et al. [83] pointed out this biocatalytic route of synthesis in 2001 using penicillin G acylase to obtain vanillyl alcohol from natural capsaicin (Scheme 22.6). As the vanillin obtained can be labelled as natural. 

Figure 10.4 Acylation of (S)-phenylglycine methyl ester in the presence of penicillin G acylase.

Enantioselective enzymatic amide hydrolyses can also be applied for the preparation of optically active organosilicon compounds. The first example of this is the kinetic resolution of the racemic [l-(phenylacetamido)ethyl] silane rac-84 using immobilized penicillin G acylase (PGA E.C. 3.5.1.11) from Escherichia coli as the biocatalyst (Scheme 18)69. (R)-selective hydrolysis of rac-84 yielded the corresponding (l-aminoethyl)silane (R)-85 which was obtained on a preparative scale in 40% yield (relative to rac-84). The enantiomeric purity of the biotransformation product was 92% ee. This method has not yet been used for the synthesis of optically active silicon compounds with the silicon atom as the center of chirality. 

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