Analysis of the trypsin from Antarctic krill (Euphausia superba) reveals enhanced enzymatic cold-adaption in low-temperature environments

Abstract Antarctic krill ( Euphausia superba Dana, 1850), a dominant psychrophilic species in the cold Antarctic and sub-Antarctic marine environments, relies on specialized enzymatic mechanisms for survival in low temperatures. Trypsin, a key enzyme in food digestion and zymogen activation, plays a crucial role in cold adaptation. However, the molecular mechanisms underlying this adaptation remain poorly understood. This study characterized trypsin genes from E. superba and warm-adapted crustaceans, revealing differences in structure and sequence. Activated trypsin from E. superba was expressed and purified using the E. coli BL21 (DE3) system. The enzyme exhibited optimal catalytic efficiency at 40°C, a lower temperature compared to trypsin from warm-adapted species like Penaeus vannamei . Multiple sequence alignments revealed conserved active and substrate-binding sites, alongside distinct residue proportions in E. superba trypsin relative to warm-adapted counterparts. Structural predictions showed a higher proportion of random coils and increased flexibility in the E. superba trypsin protein, supporting its cold-adaptive functionality. PAML analysis identified potential selection sites within the E. superba trypsin gene. Site-directed mutagenesis further suggested that strong selection pressure at these sites contributes to its adaptive evolution in cold environments. This research provides insights into the cold-adaptation mechanisms of E. superba trypsin, advancing our understanding of enzymatic functionality in extreme environments. These findings may also support the development of recombinant cold-adapted enzymes for biotechnological applications.