Investigation of various AprX endopeptidases in milk: heat stability, substrate selectivity and a novel sample preparation method for subsequent detection

AprX, metallopeptidases secreted by Pseudomonas spp. in milk, are described for their heat stability, especially during UHT treatments. Thus, they are described for their involvement in protein destabilization of shelf-stable milk products. Results showed temperature inactivation at 55 °C enhanced inactivation of most AprX compared to other temperatures. Further, AprX's half-life at 55 °C was 7.7–14.4 min. Substrate selectivity from eight Pseudomonas AprX was investigated using para-nitroaniline-substrates. The preference for Arg or Ala at P1-position at the cleavage site was linked to the AprX sequence identity. Shelf-life experiments at 20 °C determined a critical endopeptidase activity threshold of 0.1 pkat mL⁻¹ correlated with the protein destabilization for three months. Detection of such low endopeptidase activities requires a sample preparation and concentration method, which was developed. The two-step preparation method removed milk proteins and concentrated AprX 10-fold allowing a 60 ± 0.3 % recovery and precise quantification (coefficient of variation: 8.51 %).