Biochemical Characterization of a Heat-Resistant κ-Carrageenase Capable of Tolerating High Temperatures up to 100 °C

κ-Carrageenase plays a crucial role in the high-value utilization of carrageenan. Heat resistance is a key factor in the practical application of κ-carrageenase, as carrageenan exhibits gel-like properties. Previous studies have shown that the C-terminal noncatalytic domains (nonCDs) can affect the thermostability of κ-carrageenases. In this study, we expressed and characterized a κ-carrageenase, MtKC16A, which contains three nonCDs, from Microbulbifer thermotolerans. MtKC16A has the highest activity at 80 °C and pH 7.0. Surprisingly, it exhibits excellent heat resistance, with 71.58% relative activity at 100 °C and still retains over 50% residual activity after incubation at 100 °C for 60 min. Additionally, MtKC16A has been shown to have a dual substrate hydrolysis activity. It can degrade κ-carrageenan to produce highly single Nκ4 and degrade β/κ-carrageenan to produce Nκ2 and desulfated Nκ4 DA-G-DA-G4S, suggesting its potential in producing κ- and β/κ-hybrid oligosaccharides. Furthermore, we found that the unknown function domain (UNFD) in MtKC16A plays the most vital role among the three nonCDs. When this UNFD is truncated, the resulting mutants completely lose their catalytic ability at 100 °C. Finally, by introducing this UNFD to the C-terminal of another κ-carrageenase CaKC16B, we were able to improve its heat resistance at 100 °C.