For biotransformation experi ments, 1 mM four coumaric acid, caff

For biotransformation experi ments, one mM four coumaric acid, caffeic acid or ferulic acid in 200l DMSO was added to E. coli cultures at an original OD of 0. one 0. two. Cultures grew for an extra 48 hrs at thirty C before harvest and extraction. Development 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. one 0. 2, split into three separate 500 mL flasks, every single containing 200 mL of culture, and supple mented with 1 mM 4 coumaric acid. Development was contin ued for an additional 48 hrs at thirty C and OD was monitored at 600 nm. 1 mL samples had been eliminated peri odically for examination and quantification of four coumaric acid and response solutions. Extraction of culture media Past function had proven that less than 5% of goods and phenylpropionic acids were identified inside the cell pellets.
thus only culture media was extracted. For extraction, 1 mL from the culture was centrifuged at maxi mum pace to pellet cells. Media was decanted to a fresh one. 5 mL microfuge tube and selleckchem the pH was adjusted by addition of 50l hydrochloric acid. fol lowed by overnight freezing at 20 C. Tubes had been thawed at area temperature and extracted twice with an equal volume of ethyl acetate. Ethyl acetate was dried under nitrogen gasoline, as well as the dried residue was resus pended in 100l methanol. All samples were stored at twenty C just before HPLC and LC MS examination. HPLC examination 10l of extract was applied to a Zorbax RX C18 column working with an Agilent 1100 HPLC technique outfitted having a photodiode array detector. Resveratrol and ferulic acid derived items 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 which has a movement fee of 1. 0 mL min. Piceatannol was eluted by using a flow rate of 0. five mL min applying the following ailments from 0 ten min 75 25 A B, followed by selleckchem TWS119 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 instances and UV Vis spectra of typical compounds and mass spectrome try out. For quantification of products, normal curves had been constructed by plotting peak parts of acknowledged quantities of stilbene specifications. LC ESI MS examination LC Mass spectrometry was carried out that has a LCQ mass spectrophotometer equipped that has a Zorbax RX C18 column and eluted at one. 0 mL min underneath isocratic circumstances of water methanol. Mass fragmentation spectra of standard compounds along with the extracted compounds were monitored inside a mass variety of m z 100 500 using a nega tive electron spray ionization interface as described previously.

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