Improving the low-temperature activity of ꞵ-mannanase based on its structural information

Cold adaptation of enzymes is a favorable character for their industrial application because it can reduce heating costs to reach the optimal temperature for enzymatic activities. Structural feature of cold-adapted enzymes is regarded as high flexibility. On the other hand, the high flexibility can cause structural instability, resulting in lower enzymatic activity. Therefore, the good balance between structural flexibility and stability is crucially important for industrial application of enzymes. We focused the regulation of salt bridges in protein structures, which contribute to structural stabilities. Endo-1,4-ꞵ-mannanase have been used for various bioprocesses such as bleaching of softwood pulps, declining viscosity of feeds and foods, and clarifying beverages. We have already reported that the earthworm Eisenia fetida has some cold-adapted enzymes, however, mannanase from E. fetida (Ef-Man) showed only weak activity at lower temperatures (~30 ºC). We determined the crystal structure of Ef-Man at 1.7 Å resolution. The overall structure of Ef-Man is similar to those of the glycoside family 5 family proteins, and tertiary structures around the active site are conserved among endo-1,4-ꞵ-mannanase families. Ef-Man has 12 salt bridges, and we focused three salt bridges which have pairwise and bifurcated hydrogen bonding interaction between side chains. We designed and produced three Ef-Man mutants, R125K, R213K, and R302K. Enzymatic activities in all mutants improved at lower temperatures, especially the activity of R302K was about three times higher than that of wild type at 20 ºC.AsCA2018/CRYSTAL3