Studies On The Enzymatic Properties Of Fucoidanase From Mutagenized Paenibacillus Agaridevorans And The Anticoagulation Of Its Enzymatic Products

Fucoidan has a variety of biological activities.Low molecular weight fucoidan has attracted much attention as it could be easily adsorpted.The biological enzyme method was believed to be the most promising degradation method for the preparation of low molecular weight fucoidan sulfate.In this study,the crude fucoidan sulfate extracted from Kjellmaniella crassifolia was used as raw material,two strains of Pseudoalteromonas 2P5 and Paenibacillus agaridevorans 0p16 obtained from sea mud along Pikou sea coast of Dalian which could produce fucoidanase,were mutagenized to produce the enzyme and its enzymatic properties were studied.In order to obtain strains with high enzyme activity,UV mutagenesis experiments were carried out on Pseudoalteromonas 2p5 and Paenibacillus agaridevorans 0p16,respectively.The enzyme activity of Pseudoalteromonas after UV mutagenesis reached 258.72 U/mL,which was 1.58 times higher than that of the original strain;The enzyme activity of Paenibacillus agaridevorans after UV mutation was 283.5 U/mL,which was 1.36 times higher than that of the original strain.The enzyme activities of the two strains were significantly improved after UV mutation,and both strains had genetic stability.The mutant strain of Paenibacillus agaridevorans was determined for following study by comparing the degradation effect of fucoidanase produced by two mutant strains on fucoidan extracted from Kjellmaniella crassifolia.The crude fucoidanase was made by acetone precipitation method by fermentation of mutant strain of Paenibacillus agaridevorans.And then the crude fucoidan(F)from Kjellmaniella crassifolia was degraded by the fucoidanase.The enzymatic product was separated and purified by Sephacryl S-100 gel column and four fractions of F1,F2,F3 and F4 obtained.Their molecular weights were estimated as F1 of M>10 kDa,F2 of 5 kDa<M<10 kDa,F3 of1 kDa<M<5 kDa and F4 of M<1kDa.The results of TLC analysis of four components confirmed that low molecular fucoidan sulfate oligosaccharides were produced.The further separation and purification of F4 by bio-gel-p10 polyacrylamide gel column chromatography demonstrated that fucoidan sulfate was degraded to produce oligosaccharide,indicating that the degrading enzyme extracted from the mutant strain of Paenibacillus agaridevorans had significant degradation effect.The anticoagulant activity experiments were carried out on the crude fucoidan F and the purified fraction F1-F4 by Sephacryl S-100.The results showed that the crude fucoidan F and fractions F1-F4 had anticoagulant activitits and inhibited the coagulation reaction through endogenous coagulation pathway,which allhad the significant effect on prolonging activated partial thrombin time(APTT)and thrombin time(TT)in a dose-dependent correlation.At the same concentration,the prolongation of APTT decreased with the decreases of molecular weight,and the molecular weight of F1-F4fractions had little effect on prolonging TT.The extracted fucoidanase was separated and purified by DEAE Sepharose fast flow weak anion chromatography column,and three separated components E1,E2and E3 were obtained.The component E2 was selected to study the enzymatic properties.The results showed that after the fucoidanase produced by the mutant strain of Paenibacillus agaridevorans was separated and purified by DEAE Sepharose fast flow weak anion chromatography column,the optimum reaction temperature of the enzyme was 25?,Under the condition of 30-35?,the enzymatic properties are stable,and the relative enzyme activity can be maintained at more than 91.7%;The optimum reaction pH of the enzyme is 7.0,the stability is good at pH 6.0-8.0,and the relative enzyme activity can be maintained at more than 87.18%;Different concentrations(2.5,5,10 and 20mmol/L)of K⁺,Ca²⁺,Mn²⁺and 2.5,5 and20mmol/L of Mg²⁺can promote the enzyme activity.Among them,low concentrations(2.5,5mmol/L)of Mn²⁺can significantly promote the enzyme activity,with the highest relative enzyme activity of 150%.Various concentrations of Fe³⁺,Cu²⁺and EDTA have strong inhibitory effects on the enzyme activity,with the highest Fe³⁺of 20mmol/L,and the inhibition rate of 65.91%,The inhibitory effect of Cu²⁺on enzyme activity increased gradually with the increase of concentration.The molecular weight of component E2 separated and purified by DEAE Sepharose fast flow was estimated by SDS-PAGE,The results showed that the molecular weight of purified component E2 ranges from 38 kDa to 45 kDa,and its molecular weight is estimated to be 42 kDa.