A cold-adapted subtilisin-like protease from Antarctic fungus Pseudogymnoascus sp. OUCMDZ-4032: Identification,...

Cold-water washing has garnered significant attention as an effective strategy for energy conservation and carbon emission reduction. However, most commercially available detergent proteases are derived from mesophilic bacteria, exhibiting limited catalytic efficiency at low temperatures and insufficient source diversity. In this study, a novel cold-adapted subtilisin-like protease, designated PdSP4, was screened and identified from the Antarctic fungus Pseudogymnoascus sp. OUCMDZ-4032. The active band was localized using casein zymography, and the amino acid sequence was elucidated by mass spectrometric analysis, followed by successful heterologous expression in Pichia pastoris and Escherichia coli. Comprehensive enzymatic characterization suggested that PdSP4 retained better catalytic performance toward casein at 0-30 °C than a commercial alkaline protease from Bacillus licheniformis under the tested conditions. The enzyme displays an optimal activity at pH 8.0-9.0, and maintains robust tolerance to various nonionic surfactants and common metal ions. Notably, at 8 °C, PdSP4 effectively removes blood stains from fabrics and displays a pronounced synergistic washing effect when combined with commercial detergents, indicating its strong potential for cold-water laundry applications. In addition, a structure-guided protein engineering strategy led to the construction of a L245G mutant, which further enhanced the low-temperature catalytic activity of PdSP4. Collectively, this study provides a new fungal-derived candidate protease for cold-water detergents and lays a foundation for subsequent protein engineering optimization and industrial application.