For biotransformation experi ments, one mM four coumaric acid, caffeic acid or ferulic acid in 200l DMSO was extra to E. coli cultures at an initial OD of 0. 1 0. 2. Cultures grew for an extra 48 hrs at thirty C prior to harvest and extraction. Growth and production curves Overnight culture of E. coli pAC 4CL1 pUC STS was inoculated one 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. 1 0. 2, split into 3 separate 500 mL flasks, each containing 200 mL of culture, and supple mented with 1 mM four coumaric acid. Growth was contin ued for an extra 48 hrs at thirty C and OD was monitored at 600 nm. one mL samples have been removed peri odically for analysis and quantification of 4 coumaric acid and response goods. Extraction of culture media Prior operate had proven that lower than 5% of items and phenylpropionic acids have been discovered in the cell pellets.
thus only culture media was extracted. For extraction, 1 mL on the culture was centrifuged at maxi mum velocity to pellet cells. Media was decanted to a fresh 1. five mL microfuge tube and selleck the pH was adjusted by addition of 50l hydrochloric acid. fol lowed by overnight freezing at twenty C. Tubes were thawed at space temperature and extracted twice with an equal volume of ethyl acetate. Ethyl acetate was dried beneath nitrogen gasoline, and the dried residue was resus pended in 100l methanol. All samples have been stored at 20 C prior to HPLC and LC MS evaluation. HPLC analysis 10l of extract was utilized to a Zorbax RX C18 column making use of an Agilent 1100 HPLC program outfitted using a photodiode array detector. Resveratrol and ferulic acid derived merchandise had been eluted with an isocratic mobile phase of water containing 0.
1% trifluoroacetic acid and methanol containing 0. 1% trifluoroacetic acid in the ratio of 73 27 that has a flow charge of 1. 0 mL min. Piceatannol was eluted which has a movement rate of 0. 5 mL min working with the following disorders from 0 10 min 75 25 A B, followed by selleck xl-184 a gradient from 75 25 A B to 50 50 A B in 15 minutes, followed by five min 50 50 A B. Compound peaks had been identified by comparison to retention times and UV Vis spectra of typical compounds and mass spectrome try. For quantification of products, normal curves were constructed by plotting peak regions of regarded quantities of stilbene specifications. LC ESI MS examination LC Mass spectrometry was carried out which has a LCQ mass spectrophotometer equipped by using a Zorbax RX C18 column and eluted at one. 0 mL min below isocratic problems of water methanol. Mass fragmentation spectra of common compounds as well as extracted compounds have been monitored in the mass array of m z 100 500 by using a nega tive electron spray ionization interface as described previously.