Another feeding approach that can be used is based on the direct or indirect feedback control systems for the controlled addition of nutrients. Indirect control is based on online monitoring of parameters such as pH, dissolved oxygen, CO2 evolution rate and cell concentration. Direct feedback is based on monitoring the concentration of the major carbon substrate [14] and [22].

In this work, a fed-batch bioprocess was developed, via an up-scaling of hSCOMT production. Initially, several batch fermentations were carried out, in order to establish the ideal culture conditions, Sunitinib for instance batch phase and bioreactor operation for the fed-batch fermentations. After this stage, several fed-batch fermentations with different feeding profiles were tested in order to maximize biomass production and to improve protein activity levels, without compromising cell viability. Ultrapure reagent-grade water was obtained from a Mili-Q system (Millipore/Waters). Carbenicillin disodium

salt, calcium chloride dihydrate, magnesium sulfate heptahydrate, lysozyme, cobalt(II) chloride hexahydrate, dithiothreitol (DTT), SAM chloride salt, DNase, epinephrine (bitartrate salt), disodium ethylenediamine tetraacetic this website acid (EDTA), sodium octyl sulfate (OSA), bovine serum albumin (BSA), LB-Agar, IPTG, tryptone, glycerol and propidium iodide (PI) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Potassium chloride, sodium chloride, boric acid were supplied by Fluka (Buchs, Switzerland). Sodium phosphate dibasic and potassium dihydrogen phosphate monobasic were obtained from Panreac (Barcelona, Spain). Bis-(1,3-dibutylbarbituric acid)trimethine oxonol (BOX) was obtained from Molecular Probes®, Invitrogen, part of Life Technologies (Carlsbad, CA, USA). All other chemicals were of analytical grade and used without further purification. The Champion pET101 Directional TOPO expression kit

(Invitrogen Corporation, Carlsbad, CA, USA) was used for the expression of hSCOMT on E. coli BL21(DE3) strain kindly provided by Bial (Departamento de Investigação e Desenvolvimento, São Celastrol Mamede do Coronado, Portugal). In this study, except for tryptone and glycerol concentrations, all media components for the semi-defined medium were kept constant (5.5 g/L Na2HPO4, 0.5 g/L NaCl, 1.64 g/L citric acid monohydrate, 2 g/L potassium citrate, 1.21 g/L MgSO2·7H2O, 50 μg/mL carbenicillin and 1.5 mL/L trace elements solution) for the pre-cultivations, batch, and batch phase of fed-batch experiments. The trace elements solution consisted of 27 g/L FeCl3·6H2O, 2 g/L ZnCl2, 2 g/L CoCl2·6H2O, 2 g/L Na2MoO4·2H2O, 1 g/L CaCl2·2H2O, 1.2 g/L CuSO4 and 0.5 g/L H3BO4, prepared in 1.2 M HCl. LB agar plates supplemented with 50 μg/mL carbenicillin were inoculated from a cell bank aliquot and grown overnight at 37 °C.