earticle

영어

A fucoidan-degrading enzyme activity has been produced from Sphingomonas paucimobilis PF-1 (KCTC 11130BP) using Miyeokgui fucoidan, isolated from Korean Undaria pinnatifida sporophyll, as s sole carbon source of culture medium. The enzyme was purified to an apparent homogeneity mainly by sonic disruption of cells, ammonium sulfate precipitation, DEAE-Sepharose column chromatography and chromatofocusing, with an yield of 3.2% and a specific activity of 2.143 U/mg protein. The purified enzyme (tentatively named FNase S) appeared as a single band on Native PAGE gels with a molecular mass of approximately 130 kDa. However, the SDS-PAGE gels gave 3 separate proteins with molecular masses of approx. 130 (designated as S1), 70 (S2) and 60 (S3) kDa, respectively, suggesting a multi-protein complex nature of this enzyme. The first 10 N-terminal amino acid sequences of S1 (SXPEAASLPG), S2 (SPQFDVVXIG), and S3 (SLQFDVVVIG) showed high homology (over 80-90 identity) with the N-terminal regions of ATPase, core domain (EGF 27867), carbohydrate kinase, PfkB family protein (YP003854323) and dihydrolipoyl dehydrogenase (ZP08017938), suggesting that these three proteins may delineate a new family of glycoside hydrolases. The optimum conditions for enzyme activity on Miyeokgui fucoidan were pH 6.0-7.0 and 40-45℃. The activity was stable within pH 5.5-8.0 and most stable at 40-45℃ for 1 day at pH 6.0. The enzyme was activated by Mn2+ and Na+ at the concentration of 1 mM. The enzyme was specific (almost equally active) towards Miyeokgui fucoidan, commercial fucoidan (Sigma) and alginate, but exhibited very low activity towards heparin, starch, laminarin and dextran. Apparent Km, Vmax and Kcat values for Miyeokgui fucoidan were 1.7 mM, 0.62 μmol/mlㆍmin, and 0.38 S-1, respectively. The purified FNase S depolymerized Miyeokgui fucoidan into at least more than 7 distinct low-molecular weight fucose-containing oligosaccharides, ranging from 318 to 3,312 Da, with no production of monosaccharides, suggesting that this enzyme is an endo-acting fucoidanase and may be an attractive material for not only industrial applications utilizing low-molecular weight fucooligosaccharides but also structural analysis of fucose-containing marine polysaccharides.