For biotransformation experi ments, one mM 4 coumaric acid, caffeic acid or ferulic acid in 200l DMSO was extra to E. coli cultures at an first OD of 0. one 0. 2. Cultures grew for an extra 48 hrs at thirty C just before harvest and extraction. Growth and production curves Overnight culture of E. coli pAC 4CL1 pUC STS was inoculated 1 200 into 700 mL fresh modified M9 medium containing glycerol or glucose, and supplemented with chloramphenicol and carbenicillin. The culture was grown to an OD of 0. one 0. 2, split into three separate 500 mL flasks, every containing 200 mL of culture, and supple mented with one mM four coumaric acid. Development was contin ued for an additional 48 hrs at 30 C and OD was monitored at 600 nm. one mL samples have been eliminated peri odically for analysis and quantification of four coumaric acid and response goods. Extraction of culture media Preceding operate had shown that under 5% of products and phenylpropionic acids were found within the cell pellets.
hence only culture media was extracted. For extraction, one mL of your culture was centrifuged at maxi mum pace to pellet cells. Media was decanted to a fresh one. five mL microfuge tube and Sorafenib structure the pH was adjusted by addition of 50l hydrochloric acid. fol lowed by overnight freezing at 20 C. Tubes were thawed at room temperature and extracted twice with an equal volume of ethyl acetate. Ethyl acetate was dried under nitrogen gasoline, plus the dried residue was resus pended in 100l methanol. All samples had been stored at 20 C just before HPLC and LC MS analysis. HPLC evaluation 10l of extract was utilized to a Zorbax RX C18 column using an Agilent 1100 HPLC program outfitted which has a photodiode array detector. Resveratrol and ferulic acid derived products have been eluted with an isocratic mobile phase of water containing 0.
1% trifluoroacetic acid and methanol containing 0. 1% trifluoroacetic acid within a ratio of 73 27 having a movement charge of 1. 0 mL min. Piceatannol was eluted that has a flow fee of 0. five mL min making use of the next conditions from 0 10 min 75 25 A B, followed by selleck chemicals a gradient from 75 25 A B to 50 50 A B in 15 minutes, followed by 5 min 50 50 A B. Compound peaks had been recognized by comparison to retention times and UV Vis spectra of typical compounds and mass spectrome consider. For quantification of items, typical curves have been constructed by plotting peak locations of acknowledged quantities of stilbene requirements. LC ESI MS evaluation LC Mass spectrometry was carried out with a LCQ mass spectrophotometer outfitted by using a Zorbax RX C18 column and eluted at one. 0 mL min under isocratic problems of water methanol. Mass fragmentation spectra of typical compounds as well as extracted compounds have been monitored inside a mass assortment of m z one hundred 500 using a nega tive electron spray ionization interface as described previously.