C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

kappa-Carrageenase is an important component for kappa-carrageenan oligosaccharide production. Generally, noncatalytic domains are appended to carbohydrate-active domains and potentiate catalytic activity. However, studies devoted to kappa-carrageenase are relatively few. Here, a C-terminal bacterial immunoglobulin-like domain (Big_2) was identified in kappa-carrageenase (PpCgk) from Pseudoalteromonas porphyrae. Biochemical characterization of native PpCgk and its two truncations, PpCgkCD (catalytic domain) and PpBig_2 (Big_2 domain), revealed that the specific activity, catalytic efficiency (k(cat)/K-m(app)), specific kappa-carrageenan-binding capacity, and thermostability of PpCgk were significantly higher than those of PpCgkCD, suggesting that the noncatalytic PpBig_2 domain is a multifunctional module and essential for maintaining the activity and thermostability of PpCgk. Furthermore, it was found that the mode of action of PpCgk was more processive on both the dissolved and gelled substrates than that of PpCgkCD, indicating that PpBig_2 contributes to the processivity of PpCgk. Interestingly, PpBig_2 can be used as an independent module to enhance the hydrolysis of kappa-carrageenan through its disruptive function. In addition, sequence analysis suggests that Big_2 domains are highly conserved in bacterial kappa-carrageenases, implying the universality of their noncatalytic functions. These findings reveal the multifunctional role of the noncatalytic PpBig_2 and will guide future functional analyses and biotechnology applications of Big_2 domains.

Automated summary

In this study, a C-terminal bacterial immunoglobulin-like domain (Big_2) was identified in kappa-carrageenase and found to be essential for maintaining the activity and thermostability of the enzyme. The noncatalytic Big_2 domain was also shown to contribute to the processivity of the enzyme and could enhance hydrolysis of kappa-carrageenan independently. Additionally, sequence analysis revealed the conservation of Big_2 domains in bacterial kappa-carrageenases, suggesting their universal noncatalytic functions.