Penidllin production

In this chapter, by using the examples of -lactams we have briefly examined how microbial cultures may be used to produce sufficient antibiotics to meet market demands. We have also explained how enzymes (or cells) may be used to biotransform, and thereby diversify, antibiotics. By outlining the history of penidllin production, we explained how analysis and manipulation of culture regimes may be lued to enhance the yields of antibiotics (and other secondary products). These studies led to fl e concept of directed biosynflvesis by precursor feeding. 

In pwindple, by adding derivatives of acetic add to culture media, we might be able to produce a wide range of penidllins- This strategy was adopted, eg indusion of phenoxyacetic add led to the production of penidllin V ... 

Figure 6.14 Enzymatic side chain cleavage of penicillins. 6-Aminopenicillanic acid, a valuable intermediate for the production of various semi-synthetic penicillins, can be obtained through enzyme-mediated hydrolysis of the phenylacety group of penicillin G or the phenoxyacetyl group of penicillin V. The active site of the enzyme recognises the aromatic side chain and the amide linkage, rather than the penidllin nucleus. Chemical entitles other than penicillins are therefore often good substrates, as long as they contain the aromatic acetamide moiety.

It was almost immediately recognised that the deacylated product, 7-aminocephalosporanic add (7-ACA, Figure 6.16), would be of similar importance as was 6-APA in the development of new penidllins. However, 7-ACA, the cephalosporin equivalent of 6-APA, could not be found in fermentations of Cephalosporin acremonium. In Figure 6.15 we have shown that penicillin acylase hydrolyses the acyl residue from natural cephalosporins. Up to a point this is true. These acylases will, however, only work with a limited range of acyl residues. It now seems that nature does not provide for acylases or transacylases that have the capacity to remove or change the D-a-aminoadipyl side chain of cephalosporin C efficiently in a single step. Widespread search for such an enzyme still remains unsuccessful. 

E. De Vroom, The central role of penidllin acylase in antibiotics production, Chimica Oggi 1999, 17, 65-68. 

Different strains of micro-organisms are responsible for the production of either penicillins or cephalosporins. In penidllin-produdng strains, an acyltransferase enzyme system is present which can remove tlie side chain horn isopenidllin N to give 6-aminopenicillanic acid (6-APA), and which can subsequently acylate 6-APA to generate various penidllins, the most important ones being penicillin G and V(see section 63, Table 6.2). 

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... 

The subsequent advance was rafter fortuitous and rested more with serendipity than with scientific logic. A search was made for cheaper more effective replacements for casein hydrolysate. Amon t fte tested materials was com steep liquor (C5L). CSL is a by-product of the manufacture of starch from maize kemals. Whole maize is incubated in warm water, at 50°C acidified with SO2. Thermophilic bacteria hydrolyse proteins and other components of the kemals, thereby loosening the starch granules. These are removed, leaving behind fte steep liquor which is used to treat fuifter maize kemals. Ultimately, the liquor is tcx) viscous to re-use and fte liquor is concentrated and used as cattle feed. It was ftis material tiiat was used for penidUin fermentation. Stuprisingly, fte yield of penidllin increased by a furfter 5-10 fold giving yields of 50-KX) ig ml. ... 

Kaneka Co. Ltd. started the enzymatic production of D-p-hydroxyphenylglycine (d-HPG) in 1980 in Singapore, and the immobilized D-carbamoylase reactor was introduced in this process in 1995. The annual production of d-HPG by this method is around 2,000 t. d-HPG is a starting material for the production of semisynthetic penidllins and cephalosporins, such as amoxicillin and cefadroxil. d-HPG is produced from DL-p-hydroxyphenylhydantoin (DL-HPH) by a two-step enzymatic method (Takahashi 1986). 

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