Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Abstract

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

The Endo-α(1,4) Specific Fucoidanase Fhf2 From Formosa haliotis Releases Highly Sulfated Fucoidan Oligosaccharides

“…Fhf2 and Fhf2∆484 were expressed in E. coli BL21 (DE3) harboring the Pch2 (pGro7) plasmid (Takara Biolabs, Göteborg, Sweden) and purified on Ni 2+ Sepharose resin (G.E. Healthcare, Chicago, IL, United States) as previously described for Fhf1 (Vuillemin et al, 2020). The purified proteins were desalted on PD-10 desalting columns (G.E.…”

“…Low molecular weight fucoidans are more soluble, exhibit higher molecular mobility and diffuse more easily into the cell membrane (Yang et al, 2008;Torres and Díaz, 2020), and have generally been associated with improved anticancer activities compared to high molecular weight fucoidans (Yang et al, 2008;Cho et al, 2011;Chen et al, 2015). Due to their catalytic selectivity, enzymes are attractive as tools for gentle extraction of fucoidan, retaining the sulfations (Nguyen et al, 2020) and for selective modification of fucoidans to tailor-make sulfated, low molecular weight fucoidans having potentially high bioactivity (Silchenko et al, 2013(Silchenko et al, , 2018Cao et al, 2018;Vuillemin et al, 2020;Zueva et al, 2020).…”

“…Currently, 47 putative fucoidanases are classified in family GH168, and only one of these enzymes has been characterized, while 28 putative fucoidanases have been identified in GH107 with only six of them being characterized. Five GH107 fucoidanases, FWf1 and FWf2 from Wenyingzhuangia fucanilytica CZ1127 (Zueva et al, 2020), FFA1 and FFA2 from Formosa algae KM3553 (Silchenko et al, 2013(Silchenko et al, , 2017a, and Fhf1 from the marine bacterium Formosa haliotis (Vuillemin et al, 2020) have all been characterized, but not yet been included in the CAZy database.…”

“…This is notably the case for the recombinant fucoidanases MfFcnA, Fda1, and Fda2 from Alteromonas sp. SN-1009 and most recently Fhf1 (Colin et al, 2006;Cao et al, 2018;Vuillemin et al, 2020).…”

A novel thermostable prokaryotic fucoidan active sulfatase PsFucS1 with an unusual quaternary hexameric structure

“…Insight into these enzymes is a prerequisite for solving the puzzle of how bacteria degrade macroalgal polysaccharides and notably provide an important foundation for developing targeted, selective fucoidan modification processes to study and enhance specific fucoidan bioactivities. A few endo-acting fucoidanases that specifically catalyze cleavage of α-1,4 fucosyl 12 – 14 or α-1,3 linkages 15 , 16 , have been described, while two fucoglucuronomannan specific endo-fucoidanase lyases have also been identified 15 .…”