The results showed that all three specific find more growth rates tested yielded approximately the same maximum

ODs (40–50), which were also similar to those obtained with the constant feeds. In these experiments, glycerol concentrations were generally high from the start of the feeding, due to higher feeding speeds, indicating that cells are not able to exhaust all the glycerol added to the culture medium. Taking into account the results of both feeding profiles, the selected feeding profile for hSCOMT induction fermentation was a constant feed of 1 g glycerol/L/h. As mentioned above, for the final fermentations a constant feed of 1 g glycerol/L/h was used, with a higher (50 g/L) initial concentration of tryptone in order to compensate the possible tryptone limitation during the fed-batch phase. All other bioprocess parameters remained unaltered. Firstly, a fermentation without induction was performed, in order to determine the starting point of the stationary phase with this new medium formulation, and consequently the start of the feeding (Fig. 5). As seen in Fig. 5, the stationary phase was reached at about 8 h into the fermentation, and that was the time chosen to initiate the feeding. However, and since there was no significant increase in cell growth after this point, we decided to initiate the feeding 1 h earlier (at 7 h) in the subsequent experiment,

with IPTG induction. The induction was carried out 1 h after starting of the feeding, for 4 h. In this fermentation, glycerol quantitation assays were carried out as mentioned

above Selleckchem Z VAD FMK and as expected, glycerol consumption profile was very similar to the previous assays carried out with this feeding profile, however in this case, glycerol concentration was low just from the beginning, and after 2 h of feeding, the concentrations remained the same in both replicates (data not shown). Cytometry assays were carried out as explained above, and the results for these fermentations can be seen in Fig. 6 (only for the first replicate). As the results Ixazomib show, the percentage of viable cells at the end of the fermentation are relatively high, between 84% and almost 90%. For the enzymatic assay, samples were taken every 2 h after induction (until 6 h of induction), and treated according to the method described in Section 2.2.3. Specific activity results are plotted in Fig. 5, and as we can observe an increment in activity is achieved during 6 h after induction from 56 nmol/h/mg to 442.34 nmol/h/mg. In recent years, several attempts have been performed to obtain a large quantity of active and pure hSCOMT. One of the most effective ways of enhancing recombinant protein production is the application of a fed-batch process, which highly increases cell density and, subsequently, protein production. In this work, a fed-batch bioprocess was developed for hSCOMT biosynthesis.