Studies On The Properties Of Fucoidan Degrading Enzyme From Vibrio Alginolyticus And Its Anticoagulant Activity Of Enzymatic Products

Fucoidan is a heterocyclic polysaccharide with variey of physiological activities extracted from brown algae.Low molecular weight fucoidan oligosaccharides prepared by biodegradation are attracted great attention for their easily absorption and variety of physiological activities.In this study,the crude enzyme was prepared by fermentation of Vibrio alginolyticus3W24 from the sea mud of Pikou,Dalian,when fucoidan extracted from Kjellmaniella crassifolia taking as the sole carbon source.The crude enzyme solution was separated by DEAE Sepharose Fast Flow weak anion exchange chromatography column to obtain three fractions E1,E2 and E3.E3 which had the highest enzyme activity was then analyzed by SDS-PAGE polyacrylamide gel electrophoresis and the molecular weight of E3 was estimated to be about 43 k Da and 60 k Da.The results of the enzymatic properties study showed that the optimum reaction temperature of E3 fraction was 30?,and the relative enzyme activity remained above 80%in the range of 30-40?.The optimum p H was 8.0,and the stability of the enzyme was best at p H=6.0.When Mg²⁺at concentrations of 2.5,5,10 and 20 mmol/L could promote the enzyme activity of the degradive enzyme,when Mn²⁺at concentrations of2.5,5,10 mmol/L could promote the enzyme activity of the degradive enzyme.The relative enzyme activity was raised to 185.19%when the concentration of Mn²⁺at 5 mmol/L.Cu²⁺and Fe³⁺at concentrations of 10 and 20 mmol/L also could promote the enzyme activity of degradative enzymes,while Ca²⁺and EDTA at concentrations of 10 and 20 mmol/L significantly inhibited the activity of degradative enzymes.The crude fucoidan was purified by DEAE Sepharose Fast Flow weak anion excha-nge chromatography column to obtainfour fractions of F0,F1,F2 and F3.Fraction F3was then degraded by the crude enzyme at the optimized enzymatic conditions.And the enzymatic product was purified by Sephacryl S-100 gel column to obtain four fractions,which named as SF1(above 10 k Da),SF2(5-10 k Da),SF3(1-5 k Da)and SF4(belo-w 1 k Da).The further separation of Low molecular oligosaccharide made by TLC sho-wed that the molecular weights of four oligosaccharide fractions obtained from the enz-ymatic digestion products purified by Sephacryl S-100 were significantly decreased,SF1having a relatively large molecular weight and SF2,SF3 and SF4 having smaller mol-ecular weights.The anticoagulant activity in vitro of the fucoidan fraction F3 and purified fractions SF1,SF2,SF3 and SF4 were determined by using activated partial thromboplastin time(APTT),prothrombin time(PT)and thromboplastin time(TT)as indicators.The findin-gs showed that the fucoidan fraction F3 and four low molecular weight oligosaccharide fractions could significantly prolong the APTT and TT times with the increases of the concentrations.The Low molecular oligosaccharide SF3,SF4 had none dominance com-pared with those results of fucoidan F3.This may indicated that exogenous pathways contributed to the anticoagulant activity of fucoidan.