Figure 21 SDS PAGE Gel PnGOx after IEX
By using the Online Tool “Compute pI/Mw” from the SIB ExPASy Bioformatics Resources Portal, the molecular weight of PnGOx was calculated from its amino acid sequence to be 64.5 kDa.
Considering possible additional glycosylation and therefore a slightly increased molecular weight of the protein, the strongest band on the SDS Gel (Figure 21) at around 65 to 70 kDa could be identified as a PnGOx subunit. Nevertheless the SDS gel showed several other bands, which indicate impurities caused by other proteins. To reach a higher degree of purity it might be advisable to subject the P. pastoris supernatant to an additional HIC purification step prior to IEX purification as proposed by Gao, Courjean and Mano in 2009. This might in turn increase the specific activity of the enzyme solution and therefore improve the crosslinking performance.
5.3.1.1.1 PcPOx - purification
After cell harvesting by centrifugation and cell disruption with a French pressure cell press, PcPOx was purified by one-step immobilized metal ion affinity chromatography. The buffer exchange after purification was done with 50 mM sodium phosphate buffer at pH 6.5. As the activity assay for the small scale PcPOx expression was carried out exceptionally at pH 6.5, the data given in the purification table below are converted to activity at pH 5.5 in order to provide comparability of small scale to large scale PcPOx expression (see Table 36). The total enzyme activity after completion of the purification step and after buffer exchange was 1636 U.
Table 35 PcPOx purification table, small-scale expression Purification
step
Volume (mL)
Volumetric activity
(U/mL)
Specific activity (U/mg)
Protein conc.
(mg/mL)
Total activity
(U)
Total protein
(mg)
Purification factor
Yield (%) Crude
extract 40 55.1 0.9 61.8 2206 2470.4 1 -
IMAC 22 74.4 7.0 10.7 1636 235.4 7.8 74.2
Purification of PcPOx produced on a small scale happened without any significant losses.
Further interpretation of the purification data given in Fehler! Verweisquelle konnte nicht gefunden werden. is done in comparison to large-scale expressed PcPOx (see 5.3.2.1).
5.3.2 Expression on a large scale for constructive rheological tests and baking trials 5.3.2 Expression on a large scale for constructive rheological tests and baking trials 5.3.2.1 Expression in E. coli in an applikon® fermenter for baking trials (Batch)
E.coli B230 (from the culture collection of the BOKU food biotechnology laboratory; already carried the pET21a+-PcPOx construct) were cultivated in overall 30 L liquid medium in an applikon® bioreactor. PcPOx expression was induced with lactose and the fermentation operated as a batch culture. Thus, a larger amount of PcPOx was produced for subsequent baking trials at the Institute of Food Technology. The fermentation finally yielded a total biomass of 600.8 g, a total protein amount of 18.5 g and a total PcPOx activity of 24490 U in the crude extract.
Comparing specific activities of crude extracts allows to evaluate the small scale and large scale expression process: Specific activities of both crude extracts were about 1 U/mg. Different agitation, medium and inducer may explain minor differences between the small- and large-scale fermentation. When compared to the specific activity of the PcPOx crude extract from Pisanelli
et al. (2009), which was 2.77 U/mg, those of PcPOx found within both crude extracts are slightly lower. Methodological differences may be the explanation for those discrepancies. Briefly, expression in an applikon® fermenter with a more precise control of temperature and pH did not result in a higher fermentation yield than expression in a shaking flask.
5.3.2.1.1 PcPOx purification
After cell harvesting by centrifugation and cell disruption with a Homogenizer, PcPOx was purified by one-step immobilized metal ion affinity chromatography. The buffer exchange after purification was accomplished with 20 mM potassium phosphate buffer at pH 7.0. The PcPOx activities in the crude extract of the large-scale expression were obtained by summing up the activity values from all fractions of the crude extract, which had been subdivided into parts for cell lysis. The total enzyme activity after completion of the purification step, buffer exchange and sterile filtration was 11742 U.
Table 36 PcPOx purification table, large-scale expression
Purification step Volume (mL)
Volumetric activity
(U/mL)
Specific activity
(U/mg)
Protein conc.
(mg/mL)
Total activity
(U)
Total protein
(mg)
Purification factor
Yield (%)
Crude extract 1830 13.2 1.3 10.1 24206 18519 1.0 -
IMAC 220 111.3 6.7 16.7 24490 3671 5.1 101.2
Cross-Flow- Filtration 1 (Buffer exchange)
500 26.6 4.2 6.3 13290 3134 3.2 54.9
Cross-Flow- Filtration 2 (Volume Reduction)
280 44.3 3.7 11.9 12393 3332 2.8 51.2
Sterile filtration 265 44.3 3.7 11.9 11742 3154 1.0 48.5
The PcPOx purification table of the large-scale expression shows a significant loss of PcPOx after IMAC; almost half of the activity was lost as the imidazole was not removed fast enough after purification, which can be concluded from a heavy drop in total activity, but not in total protein. Although the final protein preparation was more than 95% pure judging from the SDS- PAGE (Figure 22), the final specific activity was low with of 3.7 U/mg compared to 16.5 U/mg found by Pisanelli et al. (2009) It can be concluded that a large fraction of the enzyme was inactive.
Figure 22 IMAC column, loaded with PcPOx crude extract (A), SDS PAGE gel with PcPOx from large-scale expression after IMAC (B)
Figure 22 A shows an IMAC column loaded with crude extract from a PcPOx fermentation; the yellowish colour in the upper region of the column was due to the intrinsic colour of the enzyme, the bluish colour in the lower region was caused by Ni2+.
Relating to the SDS PAGE gel in Figure 22 B, the purity of PcPOx from large-scale expression was satisfying as only minor impurities from 50 to approximately 55 kDa were visible. Pisanelli et al. (2009) found a molecular mass of 65 kDa for PcPOx, which complied with the strongest band on the present SDS PAGE Gel. The produced protein could therefore be verified as PcPOx and the purity of the protein solution could be assessed as suitable for its intended application.