Study Of Purification, Characteristic And Application Of Fucoidanase From Flavobacteriaceae Sp. CZ1127

Fucoidan is an important component of sea cucumber body wall, and it possessesa series of biological activities which are closely related to its structure. Enzymaticdegrading is the ideal method for polysaccharide degradation. We have isolated amarine bacterium strain which belongs to family Flavobacteriaceae and was namedas CZ1127, and it can secret intracellular fucoidanase which can degrade many kindsof sea cucumber fucoidan. On this basis, this paper studied the purification,characteristic and application of the fucoidanase from CZ1127.At first, the quantitative method of fucoidanase is established.375μL of20mmol/L Tris-HCl solution （pH7.2） supplemented with0.3mol/L NaCl and0.055%（wt/vol） sea cucumber fucoidan are first added into a centrifuge. Then, add375μLenzyme solution into the same centrifuge. The mixture is heated at28℃for30minafter shaking. After that,250μL pHBH solution is added to the mixture, and heated at100℃for5min, and then the absorbance of the mixtureis measured at415nm aftercooled rapidly. The enzyme activity can be calculated according to the amount of thereducing sugars （umol） in one time unit （min）. The detection limit of this method is0.048mU which is1/20of that of DNS. This method has a high sensitivity andprecision and therefore can meet the need of microdetermination.The fucoidanase from Flavobacteriaceae sp.CZ1127is purified by CellufineSulfate affinity chromatograph, Mono Q5/50GL anion exchange chromatographyand Superdex7510/300GL gel filtration chromatography. Purified fucoidanaseactivity recovery is1.8%and the purification factor is1476.5. The molecular weightof the fucoidanase is46kDa measured by SDS-polyacrylamide gel electrophoresis.Results of enzymatic property study show that the optional reactive temperature ofCZ1127fucoidanase is35℃, the optional pH is6.5; it is stable at4℃, and it is stableunder an environment of pH6.5-9.0. Activity of CZ1127fucoidanase depended on theconcentration of NaCl, and the maximum activity achieved when the NaClconcentration was0.15mol/L. Also, the activity can be inhibited by Cd²⁺, Zn²⁺andHg²⁺, and promoted by Ca²⁺.The sulfatase from Flavobacteriaceae sp.CZ1127is first purified by Cellufine Sulfate affinity chromatograph and Mono Q5/50GL anion exchange chromatography.Results of enzymatic property study show that the optional reactive temperature ofCZ1127sulfatase is25℃, the optional pH is8.5, it is stable at4℃with69.6%activity left after24hours, and it is stable under an environment of pH7.0-8.5withless than10%activity left when the pH is6.0. Activity of CZ1127sulfatase does notneed to be initiated by NaCl, however, adding proper NaCl can promote the activityespecially when the NaCl concentration is0.3mol/L. Activity of CZ1127sulfatase isonly9.7%left while the fucoidanase activity is80.3%left after CZ1127intracellularenzyme is dealed with pH6.0and4℃for48hours, which can remove the sulfataseactivity effectively.Back Propagation-artificial neural network （BP-ANNS） model for predicting themolecular weight of sea cucumber fucoidan during the enzymatic degradation isestablished by studying the relationship between amount of enzyme, hydrolysis timeand the molecular weight of the enzymatic degradation products. This model can beused to predict and control the enzymatic process of sea cucumber fucoidan, which isuseful for preparation of different molecular weight fucoidan. The BP-ANNS hassingle hidden layer, the input value is amount of enzyme and enzymatic hydrolysistime, and the output value is molecular weight of enzymatic degradation products. Inthis model, the hidden layer has7neurons and using logsig as its transfer function,purelin is used as the transfer function of the output layer, and trainglm is used as thetraining function. The convergence step of the model is74, MSE is0.00943, and theerror between forecast data and true data is1.06%on average. Using this model, theenzymatic degradation of fucoidan can be controlled.Above all, this study built a quantitative method for fucoidanase, purifiedCZ1127fucoidanase and studied its zymologic properties, as well realize themodelization for enzymatic degradation of fucoidan. It would be beneficial to thefurther utilization of CZ1127fucoidanase and sea cucumber fucoidan degradedproducts.