关键词: 假交替单胞菌；κ-卡拉胶酶；免疫球蛋白样结构域；酶学性质；结构稳定性

Abstract: Objective: To analyze the domains of κ-carrageenase from Pseudoalteromonas tetraodonis, construct domain truncation mutants, and investigate the effect of non-catalytic domains on the enzymatic properties and structure of P. tetraodonis κ-carrageenase. Methods: Escherichia coli was used to heterologously express the wild-type (WT) κ-carrageenase and the truncated mutant GH, containing only the catalytic domain of glycoside hydrolase (immunoglobulin (Ig)-like domain-deleted). We determined the enzymatic properties of WT and GH by the 3,5-dinitrosalicylic acid (DNS) method, and studied the changes in the microscopic structure of the enzyme using molecular docking and molecular dynamics simulation. Results: Both WT and its truncated mutant were successfully expressed in vitro. The molecular mass of WT and GH were 44.0 and 35.0 kDa, respectively, and both of them specifically cleaved κ-carrageenan. Enzymatic characterization revealed that deletion of the Ig-like domain did not affect the substrate specificity, optimal reaction temperature, optimal reaction pH, or hydrolysate composition, but decreased the enzyme’s thermal, strong acid (pH 4.0) and strong base (pH 11.0) stability, as well as its substrate affinity. Molecular docking and molecular dynamics simulation analyses showed that after the truncation of the Ig-like domain, the interaction between κ-carrageenanase and its substate κ-carrageenan tetrasaccharide was enhanced, the structural rigidity of the enzyme was increased, and the cyclic structure of the F1, F3, and F5 finger regions became less flexible, and the β-sheet structure of the F2 and F6 finger regions became more flexible. These structural changes might be the cause of the improved catalytic activity and thermal stability of GH. Conclusion: Our findings on the relationship between the non-catalytic Ig-like domain and enzyme properties of P. tetraodonis κ-carrageenase provide a theoretical basis for research on the structure and function of the enzyme, as well as its application.

Key words: Pseudoalteromonas tetraodonis; κ-carrageenase; immunoglobulin-like domain; enzymatic properties; structural stability