Pro duct yields and solution secretion costs were calculated based mostly on end sample concentrations and greatest growth fee for MTPs and on concentrations of ten samples taken at different time points for bench best bioreactors, Glycogen and trehalose content material Glycogen and trehalose assays had been dependant on the technique described by Parrou et al, In short, isoa mylase, amyloglucosidase and trehalase were utilised to degrade glycogen and trehalose to glucose. The glucose that may be formed in these reactions was mea sured which has a glucose oxidase peroxidase assay, Requirements had been used to determine the glycogen and trehalose recovery, Matrix results have been excluded by applying common addition. Enzyme activity assays for malate synthase and isocitrate lyase Samples for these measurements were kept at 80 C until analysis.
A predetermined volume of cells was lyzed together with the EasyLyse cell lysis kit and the cell extract was kept at four C Isocitrate lyase assay was adopted from, This colorimetric procedure is according to the reaction of glyoxy late, a product of isocitrate lyase, with find more info phenylhydrazine. The response mixture is composed of 6 mM magnesium chloride, four mM phenylhydrazine, twelve mM L cystein, and eight mM trisodium isocitrate in a a hundred mM potassium phosphate buffer, 985 L of this mixture was additional to 15 uL of enzyme extract. Enzyme activity was measured at 324 nm at thirty C, The malate synthase assay was also adopted from, This is a colorimetric assay depending on the reaction of coenzyme CoA with DTNB, The reaction mixture of this assay is composed of 15 mM magnesium chlor ide, 0. two mM acetyl CoA, ten mM glyoxylate and 0. 2 mM DTNB within a a hundred mM Tris buffer, 900 uL of this mixture was additional to a hundred uL enzyme extract. The enzyme activity was measured at 412 nm at thirty C.
The activity was normalized towards the amount of biomass used for that assay and is expressed in umol per minute per gram biomass. GC MS analysis of amino acids The examination in the isotopic labeling of amino acids was determined by, Briefly, cell pellets, sampled at regular state had been hydrolyzed with 6M HCl at 105 C for 24 h in sealed eppendorf tubes. Subsequently these details the hydrolyzates have been dried in a Thermomixer at 90 C for no longer than 12 h. Amino acids had been extracted from your hydrolyzed pellet applying 30 uL dimethylformamide and derivatized with thirty uL N N methyltrifluoroacetamide 1% tert butyldimethylchlorosilane for 1 h at 85 C. one uL of this mixture was injected into a TRACE gasoline chromato graph linked to a DSQ mass spectrometer equipped which has a TR 1 column. The carrier gasoline was helium as well as the flow was set at 1. 5 ml. min 1 with flow mode in split handle, The oven temperature was initially stored at 160 C for 1 min and after that the temperature was steadily elevated to 310 C at a charge of 20 C.