Queen's University at Kingston

Donald Westlake

Saul Wolfe

Susan E Jensen

A process for producing unnatural penicillins and cephalosporin derivatives thereof in which peptide analogs of ACV in which the L-.alpha.-aminodipyl moiety is replaced by L-S-carboxymethyl cysteine or other obvious substituents, are reacted with cyclase, epimerase, ring expansion and hydroxylase enzymes isolated from a cell free extract of a prokaryotic organism such as S. clavuligerus. The product depends upon the presence or absence of co-factors such as ferrous ion, .alpha.-ketoglutarate, and ascorbate. In an alternative embodiment, a penicillin analog having the formula: ##STR1## may be reduced with L-cysteine to produce an analog of isopenicillin N which may be reacted with the enzyme reagent to produce the desired penicillin or cephalosporin.

Biosynthesis of unnatural cephalosporins

Enzymes extracted from a prokaryotic .beta.-lactam producing microorganism are immobilized on a support for producing desacetoxycephalosporin and analogs thereof from L-.alpha.-aminoadipyl-L-cysteinyl-D-valine and analogs thereof. The enzymes are an epimerase, a cyclase and a ring expansion enzyme extracted preferably from S. clavuligerus, S. cattleya or S. lipmanii. The support is preferably a diethylaminoethyl ion exchange resin.

Enzyme immobilization for producing cephalosporin antibiotics

A process is disclosed for converting a peptide precursor of the ACV type, in which the valine moiety may be replaced by any readily available amino acid, to an unnatural cephalosporin of the type ##STR1## where R.sub.1 is H, lower alkyl or functionalized carboxylic group and R.sub.2 is H or lower alkyl, in which the precursor is reacted with cyclase, epimerase and a ring expansion enzyme isolated from a cell-free extract of a prokaryotic organism such as Streptomyces clavuligerus. The three enzymes may be immobilized on a suitable support medium and the conversion may be carried out in a continuous mode.

Cyclase, epimerase and a ring expansion enzyme are isolated separately from a cell free extract of a prokaryotic beta-lactam producing organism to provide three separate and stable enzyme reagents for commercial production of cephalosporins from peptide precursors. Isolation is carried out by using ammonium sulfate, gel filtration and ion exchange chromatography. The enzymes may be immobilized on a suitable support and the production of cephalosporins may be carried out continuously.

Stable epimerase reagent, cyclase reagent and ring expansion reagent for

The enzymes, cyclase, epimerase and a ring expansion enzyme, are isolated separately from a cell-free extract of a prokaryotic beta-lactam producing organism. The isolated enzymes are used to produce unnatural cephalosporins from polypeptide precursors. Isolation is carried out by adding ammonium sulfate to 40% saturation to the cell-free extract to precipitate contaminating proteins, adding ammonium sulfate to 70% saturation to the resultant supernatant to precipitate the enzymes, suspending the precipitated enzymes in a buffer, separating epimerase from the suspension by gel filtration, and separating cyclase and the ring expansion enzyme from each other by ion exchange chromatography.

Growing community of inventors

Edmonton, Canada

Donald Westlake