Characterization of endolysins

The results in this chapter originate in part from a recent publication (Van Nassau et al., 2017).

3.1.1 Molecular size and protein concentration

For all endolysins used in this work, information about the stock concentrations and molecular size were provided by the supplier (see Table 16). These data were confirmed for Listeria phage endolysins by UV-spectroscopy at 280 nm and SDS-PAGE. The experimentally determined concentration of endolysin PlyP40, Ply511, and PlyP825 stock solutions were 1.02, 0.40, and 3.46 mg/mL, which was more or less in line with the supplier’s product data sheets (except for a small deviation for endolysin Ply511). The size of proteins was roughly determined to be 44.5, 43.2, and 41.1 kDa respectively (Figure 11; calculated by plotting log10 of the molecular weight versus the relative migration distance). This is somewhat higher than the molecular weight as can be calculated from the amino acid sequences (Table 16), though it does reflect the size ratio between the individual endolysins, and SDS-PAGE is known to only give a rough indication of the molecular weight.

3.1.2 Enzymatic activity

Multiple methods have been described to study peptidoglycan hydrolases for both their enzymatic as well as antimicrobial activity (Nelson et al., 2012). In the present work, the enzymatic activity of endolysins was quantified with a standardized TRA using either purified cell wall material or whole live cells as substrate (Figure 12a). This was done in equimolar amounts in the concentration range where a linear relationship between enzyme concentration and steepest slope of the lysis curve was present (Figure 12b).

Figure 11: SDS-PAGE of endolysin PlyP40, Ply511, and PlyP825. The molecular weight of the marker proteins bands are indicated in kDa.

46 RESULTS

Two different approaches have been suggested in scientific literature to calculate the enzymatic activity from the generated OD-curves (as described in section 2.2.5). In both methods, the enzymatic activity is expressed as reduction in OD per minute per mg enzyme (ΔOD600/(min × mg)). This definition of enzymatic activity only allows for an accurate comparison of enzymes with exactly the same molecular weight. Hence, endolysin’s enzymatic activity is presented as ΔOD600/(min × µM), which reflects the enzymatic activity per mole enzyme and is therefore a more accurate description.

Although the specific enzymatic activity differed depending on the calculation method used, the ratio between the activities of the different endolysins remained more or less constant. For the Listeria phage endolysins examined, the activity of PlyP40 and PlyP825 was significantly higher than that of Ply511 (as calculated by the method of Korndörfer et al., 2006; Table 20). Nonetheless, enzymatic activities seemed to be in more or less the same range, especially when compared to the enzymatic activity of S. aureus specific peptidoglycan hydrolases, where HY-100 was found to have 3 to 5-fold higher activity than HY-133 and lysostaphin, respectively.

A direct comparison of the enzymatic activity between endolysins against different species is not relevant. The enzymatic activity is defined as the reduction in OD per time-unit and as such, the substrate and unit of measurement (i.e. absorption of light by the bacterial cell suspension) are intrinsically different between species. In addition, the enzymatic activity measured by TRA was found to be highly dependent on the substrate batch, buffer system, and strain used (data not shown), which not only limits the comparison phage endolysins in this work, but is a general limitation for the comparison of endolysins against different species and within scientific literature.

Figure 12: TRA and linear relationship of PlyP40 concentration with the slope of the linear descent. (a) Decrease in OD600

of a L. monocytogenes cell suspension (TMW 2.1512) after the addition of different PlyP40 concentrations or IPB as control (raw data). (b) The slope of the region of linear descent (of the curves in a, as determined by the method of Briers et al.,

2007a) plotted against the applied PlyP40 concentration. The linear regression curve and corresponding coefficient of determination (R²) are shown.

RESULTS 47

3.1.3 HHP stability Activity post HHP treatment

Endolysin PlyP40 is relative heat stable (75% residual activity after incubating for 5 minutes at 65 °C;

unpublished work of Christian A. Lenz, Lehrstuhl für Technische Mikrobiologie). In this section, the pressure stability of these endolysins was examined. Endolysins were therefore diluted to a concentration of 1000 mM and pressurized at 600 MPa (10 min, 30 °C). Directly afterwards, a TRA with whole live cells as substrate was performed with the HHP processed as well as the untreated control sample. The enzymatic activities of PlyP40, Ply511, and PlyP825 control samples were determined at 435, 400, and 383 ΔOD600/(min × µM), respectively. The activity of HHP-treated endolysin was equal or even somewhat higher (424, 431, and 481 ΔOD600/(min × mM), respectively), which is most likely the result of high assay and sample variability. Although this shows that a HHP treatment of 600 MPa does not permanently affect the enzymatic activity, activity might change during pressure treatment.

Activity during pressure treatment

A possible change in enzymatic activity under pressure could not be measured directly during HHP processing. Instead, the reduction in OD600 of a cell wall suspension was compared after samples were incubated with endolysin for 30 min and, in this time-frame, samples were either kept at atmospheric pressure or received a pressure treated at 300 MPa for 10 min (25 °C). An up- or downward shift in the TRA curves of pressure treated samples (at equal endolysin concentrations) would indicate a positive or negative effects of pressure on the enzyme’s activity. However, Figure 13 shows that the pressure treatment hardly affected the reduction in OD at any of the concentration examined. A very small reduction might be initially visible, but this is also present in the control and is therefore either the effect of pressure on the cell wall suspension or caused by small sample to sample variability.

Table 20: Endolysin enzymatic activity as determined by TRA. The enzymatic activity of endolysins and lysostaphin was examined in IPB against L. monocytogenes strain TMW 2.1512 or S. aureus strain TMW 2.422 and either calculated by the method of Korndörfer et al., 2006 or Briers et al., 2007a. For Listeria phage endolysins, mean values ± standard deviation of three experiments are shown. Different letters within the same method denote a statistical significant difference. Values of

single replicates are shown for HY-100, HY-133, and lysostaphin.

Endolysin Specific activity (ΔOD600/(min × µM))

Method of Korndörfer et al., 2006 Method of Briers et al., 2007

PlyP40 881.5 ± 77.9^a 570.5 ± 30.1^a

Ply511 532.4 ± 12.0^b 383.2 ± 35.1^b

PlyP825 738.7 ± 99.8^a 492.3 ± 80.8^ab

HY-100 - 1274,8

HY-133 - 414,9

Lysostaphin - 238,3

48 RESULTS

3.2 The inactivation of L. monocytogenes and S. aureus by endolysin and