Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity.
Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC50 value of 154+/-9 ng mL(-1) and LOD of 7.2+/-2.2 ng mL(-1) under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mg mL(-1). The DAOCS-ELISA was found to have increased sensitivity compared to the hole-plate bioassay (10.3 microg mL(-1)). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 microg mL(-1)) compared to that of the hole-plate bioassay (16 microg mL(-1)) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays.
Stok
Jeanette E
JE
Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom. jeanette.stok@chem.ox.ac.uk
Baldwin
Jack E
JE