The Extraction And Purification Of Sulfurylase And The Modification Of Carrageenan From Betaphycus Gelatinus

Carrageenan is one kind of water-soluble sulfate polysaccharide extracted from marine redalgae and is widely used in industries. It is one of the three industry gel produces (agar,carrageenan, algin) in the world. In this work we tried to achieve higher gel strength by usingD-galactose-6-sulfurylase to modify carrageenan. We also optimized the alkali method to treatcarrageenan. Carrageenan modified by enzyme and alkali were compared with each other.The impact factors that might affect the gel strength were also studied. The results are asfollows:Firstly, the preparation of high gel strength carrageenan by D-galactose-6-sulfurylase wasinvestigated. The extraction and purification methods were studied. The optimum condition toextract the enzyme were: frozen ground method using liquid nitrogen to destroy the cells; thebest extraction buffer was Tris-HCl (pH9.5,50mM) buffer with0.5M KCl added; optimumsolid to liquid ratio was1:3. The highest enzyme activity was9.3U. After the precipitate byusing70%ethanol, DEAE Sepharose Fast Flow chromatography, and HiTrap PhenySepharose Fast Flow chromatography, the purified D-galactose-6-sulfurylase was65kDa. Theosppeticmifuicm enzyme activity was186.45U/mg, and the purity was4.93fold with a recovery of thetotal activity of18.38%. The enzyme assay fo%te, manpde rtahteu rMe nw2+asd4ea0c℃ti.v Cataund out that the optimum pH was7.0and the2+, Mg2+, Co2+, Na+and K+ion showed no sifnificant effecton the enzyme activity, while the Ba2+and Cu2+ion deactivated the enzyme activity by25%~30ed the enzyme activity by60%.0-20U enzyme were usedto modify the carrageenan. When the amount of added enzyme was20U, gel strength of thecarrageenan was elevated to1249.23g/cm2, which was8.2fold of the untreated.Secondly, the alkali treatment method was optimized. Gel strength of carrageenan treateds1bt2yre%Kn,Og oHthp t was found to be higher than that got from other two. The results got after the singlefactor expwimerasuim me10tn2et5ms.8pan7e rdga/tocurmrteh2o,wg waohsn i8alc5tests were as follows: Optimum concentration of KOH wash℃w,a as n6d.7o fpotilmd uomf t htiem nea toifv etr ceaatrmraegnete nwaans.3T.h5e h roeusrusl.t Tshhoe wgeedthat the enzyme modification is better than the alkali modification.Then the properties of the carrageenan were tested before and after desulfation. Themolecular weight of the native carrageenan and the enzyme treated carrageenan were similar,while that of the alkali treated carrageenan was apparently lower. The sulfate content wasdctreeaemdursrupcalgeefardea teitbnouyanrne2μ w6.(-5ce11ar9r.e%and18.68%in the enzyme treated and in alkali treated carrageenanrespectively. The3viscosity were40.00mPa s and21.67mPa s, respectively. The meltingr a℃5g3e)..e6Tn℃ahn ea ncinodfn r5va3er.er4td℃e ds,p tweoc hκtri-cochsac rworapegyree eo hnfi agthnhr,e ewre whciahcrehrn aa gclseooem npparnaorsve esdhd wo twhitaehtd tt hhtheea net naatzfiytvemere wasD-galactose-6-sulfurylase.Finally, the impact factors that might affect the gel strength were studied. The gel shouldbe tested between the8th and the10th hour. Gel strength was intended to be stable when theconcentration was higher than1.5%. In general, gel strength showed lower value with higher temperature. Carrageenan gel strength was stable near the moderate pH (pH6-8). The effect ofstirring time on gel strength was not significant. When0.9%KCl was added, the gel strengthreached highest.