Softening a Salt Bridge Improves the Low-Temperature Activity of a Mesophilic Enzyme While Largely Preserving Its Thermal Stability

Abstract Mesophilic enzymes are often less active at low and moderate temperatures than their psychrophilic homologues. The earthworm Eisenia fetida has cold-adapted enzymes, but the end-β-1,4-mannanase shows weaker activity at low temperatures compared with other E. fetida enzymes. Cold-adapted enzymes need flexible structures, particularly around the active site, to achieve high activity at low temperatures. Salt bridges contribute to the rigidity and stability of proteins. We focused on salt bridges, including pairwise or bifurcated hydrogen bonds between arginine and glutamic acid/aspartic acid in E. fetida mannanase. The mutation of Asp316 or Arg318 in these salt bridges increased the low-temperature activity, especially the Arg318Lys mutant, which exhibited nearly 2.5-fold higher activity than the wild-type at 10–20 °C while largely preserving its thermal stability. Thus, softening the salt bridge by the arginine-to-lysine mutation based on the tertiary structural information increased low-temperature activity while largely preserving thermal stability.