Engineering the\nNon-catalytic Domain to Enhance Catalytic\nActivity and Thermal Stability of a Nκ2-Producing κ‑Carrageenase

κ-Carrageenases play an important role in achieving\nthe high-value\nutilization of carrageenan polysaccharides. They can be used in the\npreparation of even-numbered κ-neocarrageenan oligosaccharides\nby degrading κ-carrageenan (KC). We previously identified and\ncharacterized a κ-carrageenase, CaKC16B, with high specificity\nfor producing a single κ-neocarrabiose. It can produce a single\nκ-neocarrabiose from degrading KC. However, they also exhibited\npoor thermal stability and catalytic efficiency. To improve these\nproperties, we introduced noncatalytic domains (nonCDs) from a heat-resistant\nκ-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct\nCaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of\nthem exhibited improved enzymatic properties, including optimal reaction\ntemperature, thermal stability, substrate affinity, and catalytic\nefficiency. Remarkably, the <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> values of 16BUN and 16BUNB increased by 127\nand 290 times, respectively. Importantly, the addition of nonCDs did\nnot alter the final products of degrading KC, retaining the high product\nspecificity of CaKC16B. Interestingly, the addition of nonCDs changed\nCaKC16B’s substrate specificity for hydrolyzing KC and βκ-carrageenan,\nwith mutants exhibiting higher relative activity for βκ-carrageenan.\nWe further observed through biolayer interferometry that the binding\nand dissociation process between MtUN nonCD and βκ-carrageenan\nis faster compared to that of KC. This may explain the change in the\nsubstrate specificity observed in the mutants of CaKC16B.