The phage display of Bacillus subtilis Lipase A significantly enhances catalytic activity due to altered nanoscale distribution in colloidal solution

Abstract

Screening libraries of mutant proteins by phage display is now relatively common. However, one unknown factor is how the bacteriophage scaffold itself influences the properties of the displayed protein. This communication evaluates the effect of solution parameters on the catalytic activity of phage displayed Bacillus subtilis Lipase A (BSLA), compared to the free enzyme in solution. While the pH-and temperature-range (Figure 3a, analysis of variance [ANOVA], Tukey, p > .05). This result suggests that BSLA and phage-BSLA are in nanoscale environments that respond very differently to salt, which may be at the origin of the higher apparent activity of phage-BSLA versus native BSLA. Indeed, one possible explanation for this observation is that phage-BSLA may be partially associated with the gum Arabic/ Triton-X micelles (that contain the hydrophobic substrate 4-NPO)and that BSLA presence is rather predominant in the aqueous phase where substrate concentration is much lower. As such, increasing salt concentration would drive BSLA from the water phase towards the micelles, akin to a salting-out effect. To validate this hypothesis, poly (ethylene glycol) (PEG, 8 kDa) was added to the assay buffer, owing to its known ability to exert an excluded-volume effect on proteins in solution. The addition 8 kDa PEG to the solution is expected to drive proteins and phage out of the aqueous phase (this is the premise for PEG-induced protein and phage precipitation), and potentially towards the interface of the micelles. As illustrated in Figure 3b, the activity of BSLA progressively increased to 3-fold its initial value upon addition of PEG, suggesting re-distribution of BSLA towards substrate-rich micelles. In contrast, similar to the results obtained with NaCl, the activity of phage-BSLA was not affected by the presence of PEG (ANOVA, Tukey, p > .05). These combined results suggest that the difference in activity between BSLA and phage-BSLA lies mainly in the preferential association of phage-BSLA with substrate micelles. Finally, to validate that the phage themselves were not somehow affecting activity by for example, altering the structure of the substrate-containing micelles, wild-type M13 phage was added to assay buffer. Indeed, while a rapid increase of activity of free BSLA was observed upon addition of a small amount of phage, the overall increase of activity was small (<~1.5-fold increase) and rapidly plateaued at higher phage concentration (Figure 3c).Overall, this communication illustrates that the microenvironment and stability of BSLA did not appear to be intrinsically affected by phage display, though the phage modified the nanoscale distribution of BSLA within the micellar assay buffer. As such, caution should be exerted when selecting enzymes by phage display, as it is unlikely that this effect can be eliminated by for example, adding negative selection steps. Considering the diversity of applications and properties expected from proteins of interest, the effect observed herein for BSLA may be observed for other p...