Production, Immobilization And Properties Of Fucoidanase From Pseudidiomarina Homiensis ZJCN121

In this study, many kinds of samples, including seawater, sea mud, sea rock, rottenwood, seaweed and seashells, were collected from East China Sea near the Cangnan of searegion of Zhengjiang Province, and66strains marine-derived bacteria were isolated fromthe above samples by use of spread plate dilution method. With fucoidan as sole carbonsource,5fucoidanase producing strains were obtained by primary and secondaryscreening, accounting for7.58%of total bacteria, and it was found that the rate ofmarine-bacteria producing fucoidanse isolated from rotten wood to the total of bacteriaisolated from different samples was high,25%. ZJCN121was determined as study objectfor its topmost enzyme yield of0.71IU/mL and stable fermentation capability.Morphological, physiology and biochemistry of ZJCN121were studied, and16SrDNA sequence was determined and compared with the genebank and its phylogenetic treeshowed that homology of gene order between ZJCN121and Pseudidiomarina homiensisPO-M2was98%. Because morphological and physiology and biochemistry betweenZJCN121and Pseudidiomarina homiensis PO-M2had some difference, this strain wasidentified by Pseudidiomarina homiensis variety.In order to improve fucoidanase production, the compositions of medium wereoptimized according to single factor test, the results showed that the optimized carbon,nitrogen source was complex carbon source (wheat bran: glucose) and ammonium nitraterespectively, and the concentration of inducer(fucoidan)was0.59g/L; Wheat bran, NaCland fucoidan were the main factors that influence fucoidanase production byPlackett-Burman design; the optimized medium was composed of wheat bran2.7g/L, NaCl31.25g/L, fucoidan0.59g/L by Box-Behnken central combination design. Optimization offermentation showed that the initial pH of medium was6.5; the inoculation was2%andthe volume was100mL in250mL flask. Under above conditions, ZJCN121was cultivated24h at20℃, the fucoidanase activity reached a new high,1.19IU/mL.Fucoidanase was immobilized by three carriers---chitosan, attapulgite and D101-Iresin. It was found that the immobilization effect of chitosan was better than of the others.The optimal immobilization conditions were studied by chitosan adsorption andglutaraldehyde crosslinking. The results were as follows:1g chitosan carrier was added to1.5%glutaraldehyde and crosslinking2h.22IU fucoidanase was absorbed by the carrierwith rotation speed of40rpm and immobilized fucoidanase was obtained after4h at40℃;the immobilized fucoidanase activity was2.43IU/g. The properties of the immobilized fucoidanase and the free fucoidanase were also studied. The results were showed theoptimum temperature of immobilized fucoidanase was55℃, for the free fucoidanase was50℃. Half lost temperature of immobilized fucoidanase was69℃, for the freefucoidanase was62℃. The optimum pH of immobilized fucoidanase was7.0, for the freefucoidanase was6.5. Immobilized fucoidanase and free fucoidanase was relatively stablewithin the range of pH6.0-7.0. Metal ions such as Fe2+、Cu2+and Mn2+can inhibit theenzymatic activities. On the contrary, Ca2+increases the enzymatic activities slightly. TheKm value and the maximum reaction rate of immobilized fucoidanase was7.48mg/mL and3.19g/(L·min), for the free fucoidanase was5.44mg/mL and5.94g/（L·min）.