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Expression and Biochemical Characterization of a Novel Fucoidanase from Flavobacteriumalgicola with the Principal Product of Fucoidan-Derived Disaccharide.
Qiu Y
,
Jiang H
,
Dong Y
,
Wang Y
,
Hamouda HI
,
Balah MA
,
Mao X
.
Abstract
Fucoidan is one of the main polysaccharides of brown algae and echinoderm, which has nutritional and pharmacological functions. Due to the low molecular weight and exposure of more sulfate groups, oligo-fucoidan or fucoidan oligosaccharides have potential for broader applications. In this research, a novel endo-α-1,4-L-fucoidanase OUC-FaFcn1 which can degrade fucoidan into oligo-fucoidan was discovered from the fucoidan-digesting strain Flavobacterium algicola 12,076. OUC-FaFcn1 belongs to glycoside hydrolases (GH) family 107 and shows highest activity at 40 °C and pH 9.0. It can degrade the α-1,4 glycosidic bond, instead of α-1,3 glycosidic bond, of the fucoidan with a random tangent way to generate the principal product of disaccharide, which accounts for 49.4% of the total products. Therefore, OUC-FaFcn1 is a promising bio-catalyst for the preparation of fucoidan-derived disaccharide. These results further enrich the resource library of fucoidanase and provide the basis for the directional preparation of fucoidan-derived oligosaccharide with specific polymerization.
No. 32072159 National Natural Science Foundation of China, No. ZR2020JQ15 Natural Science Foundation of Shandong Province, No. tsqn201812020 Taishan Scholar Project of Shandong Province, No. SDAST2021qt18 Young Talent of Lifting Engineering for Science and Technology in Shandong-China, No. 202002017 Post-doctoral Innovation Project of Shandong Province
Figure 1. Mining and sequence analysis of α-l-fucoidanase OUC-FaFcn1. (A) Protein domain analysis of OUC-FaFcn1. (B) Phylogenetic analysis of OUC-FaFcn1 with other α-l-fucoidanases that were recorded in GH107 family. The neighbor-joining tree was obtained using MEGA 7.0. The red star indicates OUC-FaFcn1 of this study. (C) Protein sequence alignment of OUC-FaFcn1 with FcnA from M. fucanivornans (CAI47003.1), FFA1 and FAA2 from F. algae (WP057784217.1, WP 057784219.1), Fhf1 from F. haliotis (WP 066217780.1), FWf1 from W. fucanilytica CZ1127T (ANW96097.1), Fp273, Fp277, and Fp279 from uncultured bacterium (AYC81238.1, AYC81239.1, AYC81240.1). The two catalytic residues including Asp231 and His298 were marked by a red triangle. (D) Homology modeling structure of OUC-FaFcn1. The two catalytic residues including Asp231 and His298 which are located in the catalytic pocket were marked.
Figure 2. SDS-PAGE analysis of the purified OUC-FaFcn1. Lane M, protein molecular mass marker. Lane 1, the extract of E. coli BL21 (DE3) with pET-28a (+) vector (control). Lane 2, the crude enzyme extract. Lane 3, OUC-FaFcn1 purified by Ni-affinity chromatography.
Figure 3. Biochemical characteristics of the purified OUC-FaFcn1. (A) Optimum reaction temperature. (B) Thermal stability of OUC-FaFcn1. (C) Optimum reaction pH. (D) pH stability of OUC-FaFcn1. (E) Effects of metal ions and chemicals. (F) Substrate specificity of OUC-FaFcn1.
Figure 4. HPLC analysis of degradation products that change over time and the ESI-MS spectrum of the principal product Ⅰ.
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