The results of this study were obtained from the expression of recombinant hEKL in the Pichia pastoris expression system in YPD cultivation medium consisting of different YEs, peptones and glucose suppliers as described in the section Methods. Each particular experiment was carried out at least 4 times. After the expression period, the specific hEKL activity was measured in all media combinations. The results showed a significantly variable cleavage profile (Fig. 1), which indicates that the composition of media ingredients differs depending on the supplier. Trx-DCD1 with a linker recognition sequence for enterokinase (DDDDK↓X) was used as a substrate. We applied the EKMax™ enterokinase as a standard for the experiments as stated in Methods section, and based on the number of experiments, it was determined that 55% substrate digestion (Trx-DCD1) represents 1 unit of the commercial enzyme. From the cleavage percentage of the individual samples, it was possible to calculate the number of enterokinase units in each medium per defined volume, in other words, the enzyme activity. The results from this experiment were used for comparative studies.
The comparison of the peptones is shown in the Fig. 2. Each peptone used in the common media formulation had apparent impact on the level of expressed hEKL. The graph shows the level of enzyme activity relative to the peptone. The enzyme activity of hEKL expressed in media with peptone from Sigma-ALDRICH reached relatively a very low activity level compared to media with peptone from HIMEDIA. The result showed that enterokinase activities in media with peptone from HIMEDIA, SERVA and Sigma-ALDRICH were 0.7200, 0.3833 and 0.2397 U/ml, respectively. There was as much as a 3-fold difference between the average of the highest and the lowest measured enzyme activity. In summary, the media with HIMEDIA peptone showed a 3-fold higher enzyme activity of hEKL than the media with Sigma-ALDRICH peptone.
The differences in chemical composition in YE had also apparent effects, similarly as it was for peptone, on the enzyme activity in media. After evaluating the cleavage profile, the results showed that hEKL activities in cultivation media composed of yeast extract from different suppliers (Fig. 3) were 0.5760, 0.4441 and 0.3866 U/ ml for IMUNA, Sigma-ALDRICH, and BIOLIFE, respectively. The individual combinations varied, but ultimately yeast extract alone had the least effect on enterokinase activity in media. The comparison of averages of the lowest and the highest enzyme activities represented a 1.49-fold difference. The results indicate that the IMUNA YE is the most beneficial for enterokinase production among these three YEs.
Figure 4 shows the level of the enzyme activity of the EK with respect to the used glucose in the media. The enzyme activity in media with glucose from SERVA supplier was 0.5277 U/ ml, CENTRALCHEM (D-glucose anhydrous) 0.4472 U/ ml, CENTRALCHEM (D- glucose hydrate) 0.4182 U/ ml and Applichem 0.2016 U/ ml, on average. From these results, we concluded that glucose from Applichem had the most significant effect on enzyme activity, with activities in some cases ranging up to 4 times lower. Other media composed of glucose from other suppliers had a relatively comparable profile. The difference between the lowest and the highest average activities, in the case of glucose, was 2.62-fold.
The last graph shows the individual media sorted from the lowest to the highest activity levels (Fig. 5). The results varied significantly not only depending on one component, but on the combination of all three components of the YPD cultivation medium. The final analysis showed a 5.87-fold difference between the best and the most adverse combinations in terms of EK activity. After the comparative studies were conducted, it was possible to determine that the worst combination consisted of YE from BIOLIFE, peptone from SERVA and glucose from Applichem, and the most beneficial combination consisted of YE from IMUNA, peptone from HIMEDIA and glucose from SERVA.
In our next step, we proceeded to cultivation experiments in a small bioreactor. We selected two out of a total of thirty-six media composition combinations far enough apart in terms of enzyme activity in the media. After preparing the cultivation medium as described in the Methods, we set the appropriate parameters and initiated cultivation. We tried to maintain constant conditions throughout the entire process for both selected combinations and all replicates. Briefly, in the first phase of fed-batch cultivation, we tried to keep the specific growth rate close to its maximum by regulating the feeding of the feed medium manually, until we reached the maximum technical capabilities of the device. It means stirring 2000 rpm and aeration 4 vvm (volume to volume per minute) with a maximum feeding rate of 0.36–0.42 ml/ min. When the cultivation reached these conditions, we kept DOT (dissolved oxygen tension) nearly below 60%, and decreasing the feeding rate to 0.15 ml/ min. After 144 h of cultivation, the medium with produced enterokinase was separated from the biomass and analysed for cleavage profile.
For this experiment we chose media 19 and 18, not the best and worst ones, because YE from IMUNA was not available on the market yet and two relevant glucoses were disposable for our purposes in sufficient amounts for the fermentation at that moment. The difference between them in small volume experiments (2 ml) was 320%. We calculated the enzyme activity for worse combination (P1, Y1, G3) from the cleavage profile on SDS-PAGE (Fig. 6), and it was 42.14 U/ml and 15,588 U in total per fermentation. In the case of the better medium (P3, Y3, G2), the activity was many times higher, which is also evident from the result of SDS-PAGE electrophoresis (Fig. 7). The conversion, in this case, amounted to 879.04 U/ml, with a total volume of 371,544 U (Fig. 8). Ultimately, it represents almost 21-fold difference.