Abstract
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 Km, Vmax and Kcat 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.
Highlights
Fucoidans are a group of sulfated polysaccharides that exhibit various biological activities including anti-viral, anti-bacterial, anticoagulant and anti-tumoral activities [1,2,3,4]
We reported that the Miyeokgui fucoidan (MF) fucoidan is an O-acetylated sulfated galactofucan polysaccharide with a molecular mass of 1246 kDa, as characterized by various methods, such as organic elemental analysis, high performance liquid chromatography (HPLC) analysis of neutral sugars, Fourier transform infrared spectroscopy (FTIR), FT-Raman (Fourier transform-Raman), 1H and 13C nuclear magnetic resonance (NMR) [15]
All of the processes employed in the purification step may provide useful tools for the purification of those displayed on the cell surface since we previously reported that this fucoidan-degrading enzyme activity of S. paucimobilis PF-1 is observed with the harvested intact cells, implying its localization on the surface of the cell [14]
Summary
Fucoidans are a group of sulfated polysaccharides that exhibit various biological activities including anti-viral, anti-bacterial, anticoagulant and anti-tumoral activities [1,2,3,4]. It is generally believed that monosaccharide composition and sulfate contents are related to the different biological activities of fucoidans Despite their diverse pharmacologic activities, the structural variation, high molecular masses, and viscous nature of polysaccharides including marine-derived ones may limit their successful applications, especially as therapeutic agents, in terms of problems in polysaccharide standardization, solubility, and bioavailability [9,10]. Finding new fucoidanases and their subsequent kinetic characterizations would provide insight into the relationships between the structures and the biological activities of fucoidans and improved technologies for the production of industrially important bioactive fuco-oligosaccharides [11]. Enzymes having fucoidan-degrading activities have been sought for enzymatic modifications of fucoidans for the preparation of lower-molecular weight fuco-oligosaccharides. We first report on the purification and characterization of a fucoidanase (FNase S) from Sphingomonas paucimobilis PF-1, which degrades the high-molecular weight fucoidan (MF) into low-molecular weight galactofuco-oligosaccharides
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