The Studies On The Preparation,Enzymatic Properties And Characterization Of Fucoidanase From Marine Fusarium

Fucoidan is a kind of heteropolysaccharide particular to Brown seaweeds.Most of them,especially some low molecular weight fucoidans,have powerful and special biological activities,which depends on their molecular mass.Fucoidans of molecular mass ranging from 8,000～100,000 have obvious function.Some functions of the low molecular mass fucoidan are as follows:anti-coagulation of blood and/or anti-thrombus. Moreover,they can also restrain tumor from scattering and diminish inflammation. Fucoidanase can catalyse higher molecular mass of fucoidan into the lower,so the studies on fucoidanase are of very importance.A sort of microoganism,obtained from the sand from the north beach of Germany and named as Fusarium sp(LD8),was studied in the thesis.First,its ability to produce fucoidanase,which was lost after many generations,has been recovered successfully. Second,the solution containing crude fucoidanase was preparing by solid state fermentation of LD8 and extracting culture media with 0.05mol/L Na₂HPO₄-NaH₂PO₄ buffer(pH 7.0).Third,the enzyme was isolated and purified by centrifuging at low temperature,cold acetone precipitation,and column chromatography on SephadexG-100, with 22.7-fold and 23.9%of recovery.PAGE and SDS-PAGE were conducted to detect the purity and the latter to determine the molecular weight at the same time.A single band on both PAGE and SDS-PAGE shows that pretty pure fucoidanase has been obtained.Culture optimization was carried out,and the amount of the enzyme reached the maximum 48h after culture transfering,pI of the pure fucoidanase is 4.5 by IEF-PAGE, and the molecular mass of fucoidanase is about 64,000D by SDS-PAGE.The optimum pH and temperature are 6.0 and 60℃,respectively.Fucoidanase was stable in pH 6.0 buffer solution and the half-lost temperature was 38℃1h after incubating the enzyme.Metal ions such as Cu²⁺,Co²⁺,Ni²⁺,Fe²⁺can increase the enzymatic activities.On the contrary, Ca²⁺,Zn²⁺inhibit the enzymatic activities slightly.FT-IR(Fourier transform infrared spectroscopy)spectra and its derivation, deconvolution and curve-fitting methods were employed to determine the secondary structure of fucoidanase for the first time.In order to heighten analytical accuracy,the analysis of the secondary structure of fucoidanase was performed on amideâ… and amideâ…¢regions with software Peakfit4.12,respectively,which was anastomosed each other.The result demonstrated thatβ-sheet was the dominant component,and thatα-helix was the least part,whileβ-turn and random coil were between them at room temperature.Then effect of temperature on the secondary structures of fucoidanase were studied by second derivative spectra only in amideâ… band,which displayed that,below 60℃,α-helix increased with temperature rising,whileβ-turn decreased.All kinds of secondary structures of fucoidanase remained unchangeable at the range of 60℃to70℃, at which the fucoidanase was denatured by means of heating.It seemed that bothβ-sheet and random coil were independent of temperature at a certain temperature range.Fluorescence spectra of fucoidanase indicated that fucoidanase belonged to B protein,and that it contained Trp,Tyr and Phe residues.It was also studied that the fluorescence intensity of fucoidanase varied with pH.The result displayed that the intensity reached maximum at pH 6.0,that is to say,it decreased below or above pH 6.0. Synchronous fluorescence of fucoidanase was weak at or below pH 7 when△λ=60nm; Synchronous fluorescence of fucoidanase was stronger at alkaline condition than that at neutral or at acidic conditions when△λ=20nm.The results obtained from ultraviolet differential spectra of fucoidanase responsed to that from enzymatic activities affected by metal ions.Both Ca²⁺and Zn²⁺inhibit the enzymatic activities(see the above results)while they give negative absorption at about 230 nm which results fromα-helix,which is indicative of the reduction inα-helix. Instead,Co²⁺,Fe²⁺and Cu²⁺can activate the enzyme while they give positive absorption at about 230 nm indicative of the increase inα-helix.We can infer from the above results thatα-helix is necessary for the enzymatic activities.Ultraviolet differential spectra of fucoidanase with EDTA was also performed,on the basis of which we can obtain the result that fucoidanase may contain metal ion.And the binding of EDTA to the metal ion(s)result in the increase inα-helix and therefore in the increase in enzymatic activity.