Penicillin amidases/acylases

A seeond method of producing 6-APA came with the diseovery that certain mieroorganisms produee enzymes, penicillin amidases (acylases), which catalyse the removal of the side ehain fiom benzylpenicillin (Fig. 5. IB). 

The microbial sources of penicillin amidases/acylases required for side-chain removal were found and were quickly commercialised as whole-cell biocatalysts. 

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. 

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°... 

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... 

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]. 

This consideration is significant for hydrolytic reactions with hydrolases such as lipases, esterases, and amidases. These include penicillin amidases (synonymous with penicillin acylases) and cephalosporin acylases which are used for hydrolytic cleavage of penicillins and cephalosporins in thousands of tons per year [98]. These hydrolyses have to be performed at a pH-value of 8 which is close to the optimum pH of the enzyme. Lower pH-values lead to lower reaction rates and reversibility of the reaction, and hence to a significant loss in product formation. Higher pH-values are not advisable owing to the instability of the reaction partners. Moreover, addition of buffers is not accepted because of the costly removal of the buffer components. 

E1 = penicillin amidase E2 = amino peptidase E3 s penicillin acylase... 

Penicillin amidase. Penicillin acylase. 3.5.1.11 Penicillin + H(2)0 = a fatty acid anion + 6-aminopenicillanate. 

KaUenbeig A, van Rantwijk F, Sheldon R (2005) ImmobDization of penicillin G acylase the key to optimum performance. Adv Synth Catal 347 905-926 Kasche V (1985) AmpicUlin and cephalexin synthesis catalyzed by E. coli peniciUin amidase. 

Kurochkina VB, Nys PS (2002) Kinetic and thermodynamic approach to design of processes for enzymatic synthesis of betalactams. Biocatal Biotransform 20(1) 35-41 Lee SB, Ryu DDY (1982) Reaction kinetics and mechanism of penicillin amidase a comparative study of computer simulation. Enzyme Microb Technol 4 35-38 Lin WJ, Kuo BY, Chou CP (2001) A biochemical engineering approach for enhancing production of recombinant penicillin acylase in Escherichia coli. Bioproc Biosys Eng 24 239-247 Lindsay JP, Clark DS, Dordick JS (2004) Combinatorial formulation of biocatalyst preparation for increased activity in organic solvents salt activation of penidllin amidase. Biotechnol Bioeng 85(5) 553-560... 

Penicillin G acylase (benzylpenicillin amidohydrolase, also named penicillin amidase EC 3.5.1.11) is an enzyme used commercially to produce 6-aminopenicillanic add (6-APA), the most important intermediate for the industrial production of semisynthetic penicillins. This is achieved by the hydrolysis of penicillin G (for review, see Bruggink et al. 1998,0 Fig. 6.6, left column). 

The use of microbial amidases (acylases) for the production of 6-aminopenicillanic acid was revie% ed.33 An eunidase from coli shotted comparable efficacy in deacylating penicillins and cephalosporins containing the sterically similar phenylacetyl and thienylacetyl components. 

Lactams, i.e., penicillins and cephalosporins, represent the most important class of antibiotics. Penicillins consist of a common core, 6-aminopenicillanic acid (6-APA) and different side chains. Penicillin G (pen G), with a phenyl acetate side chain, and penicillin V (pen V), with a phenoxyacetate side chain, are fermented from the fungus Penicillium chrysogenum all the others are produced from 6-APA, which nowadays is produced mostly from pen G via penicillin G amidase (pen G amidase, pen G acylase, PGA, E.C. 3.5.1.11 Figure 7.33). Cephalosporins feature 7-aminocephalosporanic acid (7-ACA) or its deacetyl-form, 7-aminodesacetyl-cephalosporanic add (7-ADCA) as their common core cephalosporin C (Ceph C) is obtained through fermentation, and all the others are derived from 7-A(D)CA. 

Recently a number of enzymatic systems have been developed at several chemical companies including Upases (synthesis of enantiotrope alcohols, R-amid, S-amin), nitrilases (R-mandehc acid), amidases (non-proteinogenic L-amino acids), aspartic acid ammonia lyase (L-aspartic add), penicilin acylase (6-Aminopenicilanic acid), acylases (semisynthetic penicillins), etc.( Koeller and Wong, 2001 and references therin). 

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