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

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.     

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium,Formosa algae

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg²⁺, Ca²⁺ and Ba²⁺ cations strongly activated the enzyme; however, Cu²⁺ and Zn²⁺ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.     

The Endo-α(1,3)-Fucoidanase Mef2 Releases Uniquely Branched Oligosaccharides from Saccharina latissima Fucoidans

Fucoidans are complex bioactive sulfated fucosyl-polysaccharides primarily found in brown macroalgae. Endo-fucoidanases catalyze the specific hydrolysis of α-L-fucosyl linkages in fucoidans and can be utilized to tailor-make fucoidan oligosaccharides and elucidate new structural details of fucoidans. In this study, an endo-α(1,3)-fucoidanase encoding gene, Mef2, from the marine bacterium Muricauda eckloniae, was cloned, and the Mef2 protein was functionally characterized. Based on the primary sequence, Mef2 was suggested to belong to the glycosyl hydrolase family 107 (GH107) in the Carbohydrate Active enZyme database (CAZy). The Mef2 fucoidanase showed maximal activity at pH 8 and 35 °C, although it could tolerate temperatures up to 50 °C. Ca²⁺ was shown to increase the melting temperature from 38 to 44 °C and was furthermore required for optimal activity of Mef2. The substrate specificity of Mef2 was investigated, and Fourier transform infrared spectroscopy (FTIR) was used to determine the enzymatic activity (Units per μM enzyme: U_f/μM) of Mef2 on two structurally different fucoidans, showing an activity of 1.2 × 10⁻³ U_f/μM and 3.6 × 10⁻³ U_f/μM on fucoidans from Fucus evanescens and Saccharina latissima, respectively. Interestingly, Mef2 was identified as the first described fucoidanase active on fucoidans from S. latissima. The fucoidan oligosaccharides released by Mef2 consisted of a backbone of α(1,3)-linked fucosyl residues with unique and novel α(1,4)-linked fucosyl branches, not previously identified in fucoidans from S. latissima.     

假海源菌ZJCN121岩藻多糖酶的纯化及其性质

液态发酵培养假海源菌ZJCN121,粗酶液经过丙酮沉淀和Sephadex G-100柱层析分离纯化得到回收率为13.5%,纯化倍数为21.8,比活为20.59 IU·mg^-1的电泳纯岩藻多糖酶。采用SDS-PAGE电泳和EIF-PAGE电泳,分别测得酶的相对分子质量为60.2 kDa、等电点为4.3。该酶的最适反应温度为50℃,最适pH为6.5。其催化Fucus vesiculosus岩藻多糖的Km为5.87 mg·mL^-1,Vmax为6.11 g·L^-1·min^-1。Ca²⁺、Ba²⁺对酶稍有激活作用,Fe²⁺、Cu²⁺则强烈抑制酶活,Mn²⁺、K⁺、Zn²⁺也在一定程度上抑制酶活性,Mg²⁺对酶的作用效果不明显。     

Production of a Novel Fucoidanase for the Green Synthesis of Gold Nanoparticles by Streptomyces sp. and Its Cytotoxic Effect on HeLa Cells

Marine actinobacteria-produced fucoidanases have received considerable attention as one of the major research topics in recent years, particularly for the medical exploitation of fucoidans and their degradation products. The present study describes the optimization and production of a novel fucoidanase for the green synthesis of gold nanoparticles and its biological applications. The production of fucoidanase was optimized using Streptomyces sp. The medium components were selected in accordance with the Plackett-Burman design and were further optimized via response surface methodology. The fucoidanase was statistically optimized with the most significant factors, namely wheat bran 3.3441 g/L, kelp powder 0.7041 g/L, and NaCl 0.8807 g/L, respectively. The biosynthesized gold nanoparticles were determined by UV-vis spectroscopy and were further characterized by X-ray diffraction analysis, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis, and high-resolution transmission electron microscopy. Furthermore, the biosynthesized gold nanoparticles exhibited a dose-dependent cytotoxicity against HeLa cells and the inhibitory concentration (IC₅₀) was found to be 350 µg/mL at 24 h and 250 µg/mL at 48 h. Therefore, the production of novel fucoidanase for the green synthesis of gold nanoparticles has comparatively rapid, less expensive and wide application to anticancer therapy in modern medicine.     

Purification and Characterization of a Fucoidanase (FNase S) from a Marine Bacterium Sphingomonas paucimobilis PF-1

The Search for enzyme activities that efficiently degrade marine polysaccharides is becoming an increasingly important area for both structural analysis and production of lower-molecular weight oligosaccharides. In this study, an endo-acting fucoidanase that degrades Miyeokgui fucoidan (MF), a sulfated galactofucan isolated from the sporophyll (called Miyeokgui in Korean) of Undaria pinnatifida, into smaller-sized galactofuco-oligosaccharides (1000–4000 Da) was purified from a marine bacterium, Sphingomonas paucimobilis PF-1, by ammonium sulfate precipitation, diethylaminoethyl (DEAE)-Sepharose column chromatography, and chromatofocusing. The specific activity of this enzyme was approximately 112-fold higher than that of the crude enzyme, and its molecular weight was approximately 130 kDa (FNase S), as determined by native gel electrophoresis and 130 (S1), 70 (S2) and 60 (S3) kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of FNase S were pH 6.0–7.0 and 40–45 °C, respectively. FNase S activity was enhanced by Mn²⁺ and Na⁺ (115.7% and 131.2%), but it was inhibited by Ca²⁺, K⁺, Ba²⁺, Cu²⁺ (96%, 83.7%, 84.3%, and 89.3%, respectively), each at 1 mM. The K_m, V_max and K_cat values of FNase S on MF were 1.7 mM, 0.62 mg·min⁻¹, and 0.38·S⁻¹, respectively. This enzyme could be a valuable tool for the structural analysis of fucoidans and production of bioactive fuco-oligosaccharides.