Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

Abstract

The protease Ser2 secreted by the psychrotrophic strain Serratia liquefaciens L53, a highly proteolytic strain isolated from Brazilian raw milk was purified and characterized. Using azocasein as substrate, Ser2 exhibited activity in a wide range of pH (5 to 10) and temperature (4 to 60 °C). The optimal activity was detected at pH 8.0 and at a temperature of 37 °C. This protease, still active at 4, 7, and 10 °C, was strongly inhibited by chelating agents and by dithiothreitol, a reducing agent. These results confirmed that Ser2 belongs to the peptidase family M10 and requires Ca , Zn , and disulfide bridges for stability. This protease is able to hydrolyze three kinds of casein in the preferential order of κ→ β→ α-casein. Highly heat-stable in skimmed, semi-skimmed, and whole milk at 140°C with D-values of 2.8, 3.9, and 4.5 min, respectively, Ser2 showed a residual activity between 87 and 100 percent after heat-treatment of 65 °C for 30 min, 72 °C for 20 s, and 140 °C for 4 s that are commonly used in dairy industries. As the protease AprX that is mainly secreted by Pseudomonas genus, Ser2 could be one of the main causes of UHT milk destabilization during storage.

Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes

“…The residual activity of protease produced by P. fluorescens was 97.2% after heat treatment at 80 °C for 30 min (Zhang and Lv, 2014). The purified protease Ser2 secreted by S. liquefaciens was highly heat-stable in skimmed, semi-skimmed, and whole milk at 140 °C, with D-values of 2.4, 3.9, and 4.5 min, respectively, and the presence of milk fat increased the heat-stability of the protease (Baglinière et al, 2017). The heat inactivation of the purified protease from Chryseobacterium indologenes did not follow first-order kinetics, but showed biphasic inactivation curves, and this protease is much more heat-labile than other psychrotrophic proteases (Venter et al, 1999).…”

Purification and characterization of the thermostable protease produced by Serratia grimesii isolated from channel catfish

“…Other proteases from Bacillus spp . and S. liquefaciens also have shown increased activity in the presence of MnCl 2 . Reaction with MgCl 2 exhibited the same relative activity of 64% as in S. marcescens metalloprotease in whey .…”

“…Serratia has been known for infections in humans but certain members have also been identified as spoilage agents in foods. For example, S. liquefaciens , S. proteamaculans , and S. plymuthica RVH1 spoil tofu, milk, and carrots, respectively.…”

Application of LC‐HRMS identified marker peptides in an LC‐MS/MS method for detection and quantification of heat‐resistant proteolytic activity in raw milk

“…According to Bagliniere et al . (2017a; 2017b), both enzymes hydrolyse β‐, α s ‐ and κ‐caseins in UHT milk and produce similar peptide patterns in destabilised UHT milk. More research is needed to determine whether both spoilage enzymes produce the same marker peptides as identified in our study.…”

“…The former spoilage organism produces Ser2 instead of AprX as heat-resistant metalloprotease, but both enzymes belong to the same serralysin subfamily of bacterial zinc-metalloproteases and share a high degree of homology (Machado et al 2016). According to Bagliniere et al (2017a;2017b), both enzymes hydrolyse b-, a s -and j-caseins in UHT milk and produce similar peptide patterns in destabilised UHT milk. More research is needed to determine whether both spoilage enzymes produce the same marker peptides as identified in our study.…”