Preparation, Purification And Characteristics Of Fucoidanase From Pseudidiomarina Homiensis ZJCN121

Fucoidanase was a kind of polysaccharide hydrolases, and fucoidanase could be usedin preparation of low molecular weight fucoidan (LMWF) due to the advantage ofenzymatic hydrolysis, such as substrate specificity, higher catalytic efficiency, mildreaction conditions and less contamination, ease of set-up and work-up. Enzymatichydrolysis became a popular method in producing LMWF and substituted for chemicalhydrolysis.In addition, fucoidanase also played an important role in the study of thestructure, the relation between structure and mechanism of fucoidan. Therefore, Scientistswere interested in researchs on fucoidanase.In order to improve the ability of enzyme production, the medium compositions offucoidanase by liquid state fermentation were optimized via the method of combiningsingle factor test, Plackett-Burman design and response surface experiment. The resultsshowed that wheat bran, NaCl and fucoidan were the main factors which affecting theenzyme activity, and the optimal concentration of the above factors were determinedaccording to central composite design and response surface analysis, and the optimalmedium compositions (g/L) were finally obtained for: wheat bran2.73, NaCl31.04,fucoidan0.54, glucose0.455, NaNO32, MgSO43, and CaCl20.04, Na2HPO40.7. Thefermentation conditions were further optimized by single factor test, the results displayedthat the initial pH of medium was6.4, the inoculum age was12h, inoculum concentrationwas3%, the bacteria ZJCN121was cultivated at20℃for16h, the yield of the fucoidanasereached1.43IU/mL which was2.07times higher than that of the original conditions,Judged by dynamic parameter of fermentation process from ZJCN121, the synthesis typeof fucoidanase was identified as synchronous synthesis type.Pseudidiomarina sp. ZJCN121was cultured by liquid state fermentation under theabove conditions, and extracted by buffer solution. Crude enzyme solution was achievedby filtration and centrifugation successively, then it was separated and purified throughacetone precipitation and Sephadex G-100chromatography with a final purification of21.8-fold and a specific activity of20.59IU/mg and an overall yield of13.5%. Theacquisition was identified as pure fucoidanase by showing only one band on PAGEelectrophoresis, SDS-PAGE gel electrophoresis and EIF-PAGE gel electrophoresis, whichreached the aim of purification. The relative molecular weight of pure fucoidanase wasmeasured as60.2kDa, and its isoelectric point was4.3.The results also indicated that the optimum reactive temperature of purified fucoidanase was50℃, half-inactivity temperature was62℃; the optimum pH was6.6,and enzyme was stable from pH6.2to7.0. When the metal ion concentration was1mmol/L, the fucoidanase was lightly activated by Ca2+, Ba2+and strongly inhibited byFe2+, Cu2+. Its activity also was lightly inhibited by Mn2+, K+, Zn2+and unaffected by Mg2+.When Fucus vesiculosus fucoidan was used as the substrate, its Kmand Vmaxwere5.87mg/mL and6.11g/(L·min) respectively.