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Front Microbiol
2020 Jan 01;11:1674. doi: 10.3389/fmicb.2020.01674.
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Discovery and Characterization of an Endo-1,3-Fucanase From Marine Bacterium Wenyingzhuangia fucanilytica: A Novel Glycoside Hydrolase Family.
Shen J
,
Chang Y
,
Zhang Y
,
Mei X
,
Xue C
.
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Sulfated fucans are important marine polysaccharides widely distributed in brown algae and echinoderms, which gained increasing research interest for their various biological and biomedical activities. Fucanases could serve as tools in the bioconversion and structural investigation of sulfated fucans. A few gene-defined endo-1,4-fucanases have been characterized, while the sequence of endo-1,3-fucanase remain unstudied. Here, an endo-1,3-fucanase gene funA was screened from the genome of marine bacterium Wenyingzhuangia fucanilytica CZ1127T using transcriptomics. None of the previously reported glycoside hydrolase domains were predicted in the enzyme FunA, which hydrolyzed sulfated fucans in a random endo-acting manner. Ultrahigh performance size exclusion chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that FunA specifically cleaves α-1,3 glycosidic linkage between 2-O-sulfated and non-sulfated fucose residues. FunA exhibited transglycosylating activity with glycerin, methanol, and L-fucose as acceptors. D206 and E264 were critical for the functioning of FunA as identified by the site-directed mutagenesis. Another five homologs of FunA were confirmed to possess endo-1,3-fucanase activities. This is the first report on the sequence of endo-1,3-fucanase. The novelty of FunA and its homologs in sequences and activity shed light on a novel glycoside hydrolase family, GH168.
FIGURE 1. HPSEC-RID chromatograms of hydrolysis products obtained at different reaction times. The reaction time was labeled above the corresponding chromatogram. After 12 h reaction, the fresh enzyme was supplemented into the reaction mixture and incubated for a further 12 h to prepare the end product (24 h). The Superdex peptide 10/300 GL column was employed for examining the oligosaccharide pattern, and the TSKgel SuperAW4000 column was utilized for investigating the global profile (the insert).
FIGURE 2. The total ion chromatogram (A), mass spectrums of component I (B) and II (C) as determined by UPSEC-MS.
FIGURE 3. The 1H NMR (A), COSY (B), NOESY (insert of B) spectrum and the structure (C) of the major component in the end product of FunA. A1/A2 indicated the cross-peak between H-1 and H-2 of residue A, etc.
FIGURE 4. The UPSEC-MS analysis of hydrolysis and transglycosylating products produced by FunA in the reaction system with glycerin as the acceptor. (A) Extraction ion chromatograms; (B) MS/MS spectrum of [Fuc4S4Gl-2S-2H]2–. “Fuc” represented fucose residue, “Gl” represented glycerin residue, “S” represented sulfate group, “O” represented oxygen from the glycosidic bond. The charge number of fragment was provided by the instrumental software (Agilent Masshunter Qualitative Analysis Navigator B.08.00).
FIGURE 5. Phylogenetic analysis of FunA and its homologs. Sequences successfully expressed in the supernatant of E. coli cell lysate were highlighted: sequences exhibited activity on Ib-FUC were in red; sequences with no activity on Ib-FUC were in purple; sequences expressed in inclusion body were in yellow.
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