Unveiling AprX: biochemical and molecular mechanisms of a heat-resistant inducing Milk spoilage protease

The proteolysis of Ultra-High Temperature (UHT) milk is often linked to the proteolytic activity of AprX. This enzyme is a thermostable extracellular protease produced by Pseudomonas spp. that hydrolyzes caseins, causing milk destabilization and subsequent gelation. Since the availability of literature on the mechanisms involved in the action of AprX in dairy product spoilage is limited, this review article explores the biochemical aspects of AprX proteolytic activity, including its rules on cleavage sites in casein micelles. It discusses the aprX evolutionary aspects, regarding the conserved nature of its coding gene, the variable organization of the aprX - lipA2 operon, and the mechanisms regulating its expression. The review highlights different quantification methods, emphasizing colorimetric assays due to their simplicity. It also addresses the resistance of AprX to UHT treatments due to its structure, attributed to its structure being stabilized by divalent ions, and underscores the need for new methodologies to better understand and control AprX activity in dairy processing. The enzyme's high resistance to UHT treatments is due to its flexible structure stabilized by interactions with divalent ions, mainly zinc and calcium. Due to its impact on the milk and dairy industry, new methodologies must be investigated based on the biochemical knowledge governing the AprX molecule. This review underscores the knowledge gaps and technological challenges that hinder efficient monitoring and control of AprX in dairy processing, calling for the development of innovative approaches to mitigate its impact on product quality and safety. • AprX is a thermostable protease driving spoilage in UHT dairy products. • Cleavage specificity in caseins underlies destabilization and age gelation. • Structural and regulatory features explain AprX thermostability and activity. • Sensitive and rapid industrial-scale detection of AprX protease remains challenging. • Knowledge gaps hinder AprX control, demanding innovative mitigation tools.