Chloramphenicol → L-Phenylalanine

Participants

Reaction Steps

Both PAL and TAL activities were also investigated according to whole cell assays. Seed cultures consisting of 5 ml of LB broth containing 34 mg/L chloramphenicol were prepared of E. coli BL21(DE3) strains that were individually transformed with each of pSpalAv, pSpalNp, pSencPSm, pSpal1At, and pSpal2At. These cultures were grown for 12 hours at 30° C. while agitating at 250 rpm. 1 mL of each culture was then used to inoculate 3×250 mL cultures flasks containing 50 mL of LB supplemented with 34 mg/L chloramphenicol. All cultures were then grown at 30° C. while agitating at 250 rpm until an OD600 of 0.6 was reached, at which point the cultures were induced by IPTG addition to a final concentration of 0.2 mM. Cultures were then grown for an additional 6 h before the cells were then collected by centrifugation in 50 ml Falcon tubes for 5 min at 1400×g and washed once with pH7 PBS (phosphate buffered saline) buffer. The entire cell pellet was then resuspended in 10 ml pH7 PBS buffer before the appropriate substrate, L-phenylalanine or L-tyrosine, was added at a final concentration of 1 g/L. The results for L-phenylalanine addition are shown in FIG. 2. For all strains tested the addition of L-tyrosine did not result in the formation of p-coumaric acid, indicating that no TAL activity was displayed by these recombinant strains.

Provenance & Evidence