Expanding the application range of the κ‑carrageenase OUC-FaKC16A when preparing oligosaccharides from κ-carrageenan and furcellaran

Abstract

Carrageenan oligosaccharides are important products that have demonstrated numerous bioactivities useful in the food, medicine, and cosmetics industries. However, the specific structure–function relationships of carrageenan oligosaccharides are not clearly described due to the deficiency of high specific carrageenases. Here, a truncated mutant OUC-FaKC16Q based on the reported κ-neocarratetrose (Nκ4)-producing κ-carrageenase OUC-FaKC16A from Flavobacterium algicola was constructed and further studied. After truncating the C-terminal Por_Secre_tail (PorS) domain (responsible for substrate binding), the catalytic efficiency and temperature stability decreased to a certain extent. Surprisingly, this truncation also enabled OUC-FaKC16Q to hydrolyze Nκ4 into κ-neocarrabiose (Nκ2). The offset of Arg265 residue in OUC-FaKC16Q may explain this change. Moreover, the high catalytic abilities, the main products, and the degradation modes of OUC-FaKC16A and OUC-FaKC16Q toward furcellaran were also demonstrated. Data suggested OUC-FaKC16A and OUC-FaKC16Q could hydrolyze furcellaran to produce mainly the desulfated oligosaccharides DA-G-(DA-G4S)2 and DA-G-DA-G4S, respectively. As a result, the spectrum of products of κ-carrageenase OUC-FaKC16A has been fully expanded in this study, indicating its promising potential for application in the biomanufacturing of carrageenan oligosaccharides with specific structures.

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

“…Previous studies have shown that nonCDs PpBig_2 and FaPorS play essential roles in maintaining the specific activity, thermostability, and product compositions of κ-carrageenases PpCgk and OUC-FaKC16A, respectively. Our study also demonstrates the essential function of the Big_2 nonCD in MtKC16A.…”

“…17 Similarly, in our previous study, we found that truncation of the PorS nonCD in κ-carrageenase OUC-FaKC16A, derived from Flavobacterium algicola, also led to a decrease in thermal stability. 18 These indicated that there is a close relationship between the thermal stability and nonCDs of κ-carrageenases. This suggests a close relationship between the thermal stability and nonCDs of κ-carrageenases.…”

Comparative Study on Enzymatic Characteristics of Two κ-Carrageenases from Carrageenan-Degrading Bacterium Catenovulum agarivorans DS2

“…Likewise, the truncation of nonCDs from κ-carrageenases PpCgk and OUC-FaCK16A led to a decrease in their thermostability, substrate affinity, and catalytic efficiency. 16,17 In addition, their hydrolytic products also underwent certain changes after truncation. However, the main hydrolytic products of CaKC16AΔtoward KC and B/KC were still Nκ4 and desulfated Nκ6, DA-G-(DA-G4S) 2 (Figure S3), respectively, without any significant change, indicating different nonCDs of different κ-carrageenases might play a different role in modulating product composition.…”

“…In particular, its final product of hydrolyzing KC changed from Nκ4 to Nκ2. 17 These two studies revealed that both Big_2 and PorS nonCDs could affect the enzymatic properties, bringing us a new idea for excavating κ-carrageenases with different enzymatic properties, especially catalytic products. To validate the above mining idea, two κ-carrageenases, CaKC16A and CaKC16B, originating from the same bacterium Catenovulum agarivorans DS2, were selected.…”

“…15 Several studies demonstrated the substrate binding ability to carrageenan of these nonCDs. A Big_2 nonCD existing in κ-carrageenase PpCgk (PpBig_2) and PorS nonCD existing in κ-carrageenase OUC-FaKC16A (FaPorS) exhibited the binding ability to KC, 16,17 and a CBM nonCD existing in κ-carrageenase Cgk16A (WaCBM) exhibited the binding ability to KC and IC. 14 Due to their substrate binding ability, further explorations indicated that the nonCDs play a vital role in maintaining the thermal stability and catalytic ability of κ-carrageenase.…”