| κ-Carrageenan is water-soluble sulfated polysaccharides extracted from numerous redalgae. It corresponds to the less sulphated ideal polymer and leads to strong, elastic, andthermal hysteresis gels. These properties make it a wide range of applications as gelling,stabilizing, and viscosity-binding agents in food, cosmetics and pharmaceutical industries. Inorder to improve the gel strength of native κ-carrageenan, carrageenan is subjected to hotalkaline treatment. At present, the widely used industrial step involves soaking the red algaein a strong solution of alkali hydroxide (up to10%NaOH/KOH) at elevated temperature(70-90℃) for a few hours(up to5h). However, this process has obvious disadvantagesincluding the decrease of carrageenan yields, and generation of an alkali effluent. Recently,the eco-friendly enzyme catalysis is becoming a new trend in place of the traditionaltechnologies. D-Gal-6-sulfurylase modification could improve the gel strength ofκ-carrageenan. However, there is less evidence for this enzyme in the literature, and noresearch about the modification mechanism. In this thesis, D-Gal-6-sulfurylase was firstlyscreened from various red algae, and then purified to homogeneity by sequentialchromatography procedure. The stability and properties of this enzyme was studied.Furthermore, the purified sulfurylase was applied to produce high gel strength κ-carrageenan,and the mechanism was explored. The results are mainly as follows:At first, we screened the sulfurylase activity from various red algae, and foundEucheuma striatum was a particularly rich source for D-Gal-6-sulfurylase. Based on freshweight, the activity of sulfurylase for μ-carrageenan was10.87±0.39U/g. Moreover, thesoluble fraction showed92.6%recoverable activity, while the membrane fraction recoveredonly7.4%sulfurylase activity. The extract conditions of sulfurylase from E. striatum wereoptimized to be: frozen ground method using liquid nitrogen to destroy the cells, extractionbuffer was50mM Tris-HCl buffer (pH8.5) containing10mM2-mercaptoethanol and0.5MKCl, extraction time was6h and ratio of material to water was1:3. In this condition, theactivity of sulfurylase was6.92U/mL, and the content of protein was0.165mg/mL.The stabilization of the sulfurylase prepared from E. striatum was investigated usingselected chemical additives. The sulfurylase activity appeared sensitive to pH. The crudeextract contained10mM2-mercaptoethanol could be stored at4℃at pH7.0for a weekwithout notable loss in enzyme activity.In the presence of10%(v/v) glycerol, the stability of crude sulfurylase was not changedin10days, and maintained80%activity in20days at20°C. After lyophilization, thesulfurylase containing5%(w/v) sucrose retained98.65%residual activity, and after40daysthe activity almost unchanged at20°C. Sulfurylase was identified a thermal labile enzyme,and both of the addition of glycine and sucrose resulted in an increase in the thermal stabilityof sulfurylase, which maintained65%and60%residual enzyme activity at50℃, respectively.The lyophilized sulfurylase was purified to electrophoretic homogeneity by ammoniumsulfate precipitation, Phenyl Sepharose6Fast Flow column and DEAE Sepharose CL-6Bcolumn. By SDS-PAGE, the molecular mass of sulfurylase was estimated to be60kDa, whichwas considered to be a monomer protein. The optimum reaction pH of sulfurylase was7.0, and the optimum temperature was40℃. The Kmand Vmaxfor μ-carrageenan was4.31mM and0.17mM min–1, respectively. The sulfurylase activity was obviously inactived in the presenceof PMSF, TNBS, PCMB, which indicated that serine, lysine, cysteine was the key amino acidin active site. The enzyme activity was unchanged by EDTA, which suggested that thesulfurylase was not a metalloenzyme.The κ-carrageenan from E. striatum modified by100U of sulfurylase exhibited6.78-fold increase in gel strength (Viz.1457.4g/cm2) with a high yield of43.57%. Moreover,the gelation abilities and texture are improved. The results of decolorization of enzymemodified κ-carrageen showed that the efficiency by0.16g/mL macroporous resins D303was90.73%, retention rate of polysaccharide was85.76%and gelling strength was1103.7g/cm2,representing good decolorizing ability. On the other hand, the alkali modification process wasestablished to be: ethanol concentration was20%, alkali concentration was3%, modificationtime was1.5h, and modification temperature was75℃. The physico-chemical properties ofthe enzyme modified κ-carrageenan were comparable to that of alkali modified κ-carrageenan,and the enzyme modification had obvious advantages in environmental protection. Thedeveloped method provides an eco-friendly alternative for alkali treatment method to producehigh gelation property κ-carrageenan.According to the spectrum analysis of FTIR and1H/13C NMR, the main component ofcarrageenan from E. striatum is κ-carrageenan. Small amount of ι-carrageenan and β-carrageenan are also found. The mechanism of the improvement in gel strength ofκ-carrageenan modified by sulfurylase was explored. The results indicatied that modificationwith sulfurylase could eliminate the sulfate ester group from μ-carrageenan converting intothe corresponding3,6-anhydro ring of κ-carrageenan, and transform to helix-compatible type,furthermore, resulte in morphologically distinct product with a honeycomb network ofordered helical structures, and thus increase the gelation property. |
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