Study On Agar Enzyme-assisted Extraction And Agaro-oligosaccharides Enzymolysis Production And Agarase Immobilization

Gracilaria is rich in colloid,which is a primary material for agar extraction.Agar has wide application in food,pharmacy,daily chemistry and biochemistry.Moreover,agar can be hydrolyzed by agarase to obtain the agar-derived oligosaccharide with the great potential and value to utilize.Firstly,the process of enzyme-assisted extraction of agar from Gracilaria was studied in the thesis.Secondly,Vibrio sp.NTi,which was isolated from Xiamen mangrove soil,was used to produce agarase in this work,and then the enzymatic hydrolysis processing of agar was determined after systemic research on the rule of the effects of each factor by single factor experimental way.Then,the optimum enzymatic technology was used to prepare the agaro-oligosaccharide with different polymerization degrees.Meanwhile,the antioxidant activity and anticancer activity of agaro-oligosaccharid obtained were subsequently studied.Finally,carboxyl functional magnetic nanoparticles prepared was used to immobilize agarase,and the important factors affecting immobilization were explored,and the properties of immobilized agarase was also studied.?1?The appropriate preparation process of enzyme-assisted extraction of agar from Gracilara was studied by systemic research on the rule of the effects of each factor by single factor experimental way with gel strength as evaluation parameter.The optimal extraction process abtained in this study was:the concentration of alkali treatment is 5%,alkali treatment time is2.5 h,enzyme treatment temperature is 45°C,enzyme treatment time is 2.5 h,enzyme treatment pH is 7.0,the enzyme concentration is 4 U/mL.Based on the determination of optimal process parameters,the agar extraction process of 200 L-scale was carried out and the result proved that Gracilaria after cellulase treatment can decrease the dosage of alkali without affecting the gel strength.?2?To study dynamic changing process and technical conditions of agar hydrolysis by agarase,the content of reducing sugar was determined by a 3,5-dinitroalicylic acid assay,as well as the content of total polysaccharide was detected by phenol-sulfuric acid method.According to the ratio of reducing sugar and total sugar,the variation regularity of average polymeric degree of total sugar in reaction system has been achieved.Thus the influence of enzymatic hydrolysis affected by the reaction factors was abtained.The optimum condtions for agar hydrolysis by agarase which came from marine Vibrio sp.NTi were achieved by a series of single-factor experiments.The results showed that the concentration of reducing sugar reached 2.18 g/L and the average polymetric degree fell to 3,when substrate at the concentration of 12 g/L was dissolved in buffer solution at pH 7.0,adding 25%enzyme dosage and the agar hydrolysis was carried out in a shaking bath at 40°C and 120 rpm for 90 min.In addition,the optimal synthesizing parameters mentioned above were verified in a way of continuous and stable operation for 20 L tank magnified experiment.Furthermore,the enzymatic hydrolysate was identified by thin-layer chromatography and it was shown that the intermediate products mainly include neoagarobiose,neoagaroetraose and neoagarohexaose.The end product of degradation is neoagarobiose after a 48-hour period enzymatic hydrolysis process.?3?The antioxidant activities of agaro-oligosaccharide with different polymerization degrees were evaluated by DPPH,ABTS,·OH,·O^2-radical scavenging activites and Fe³⁺reducing activites.The IC₅₀ of agaro-oligosaccharide with average polymeric degree 3,4,5 and BHT against DPPH radical were 14.29 mg/mL,6.65 mg/mL,8.94 mg/mL?0.02 mg/mL,respectively,while the IC₅₀ of agaro-oligosaccharide with average polymeric degree 3,4,5 and BHT against ABTS radical were 9.07 mg/mL,3.04 mg/mL,4.10 mg/mL?0.04 mg/mL.In addition,the IC₅₀of agaro-oligosaccharide with average polymeric degree 4,5 and Vc against·OH radical were11.58 mg/mL,11.66 mg/mL,3.10 mg/mL,the agaro-oligosaccharide with average polymeric degree 3 had no activity of scavenging hydroxyl free radical.Futhermore,the IC₅₀0 of agaro-oligosaccharide with average polymeric degree 4,5 and Vc against O^2-radical were 75.84mg/mL,24.16 mg/mL,21.38 mg/mL,0.068 mg/mL.the reducing capacities of them were as follows in order:BHT>DP3>DP4>DP5.The antimicrobial activities of agaro-oligosaccharide with average polymeric degree against three bacterial strains and three funguses was determined by bactericidal test.The antimicrobial experiments showed that the agaro-oligosaccharide with average polymeric degree had no bacteriostasis effect.?4?A novel and efficient strategy for agarase immobilization was developed with carboxyl-functioned magnetic nanoparticles as supports.The carboxyl-functioned magnetic Fe₃O₄ nanoparticles were first prepared by co-precipitation before agarase was covalently immobilized onto the nanoparticles,with glutaraldehyde as the activation agent.The obtained particles were then characterized by transmission electron microscopy,X-ray diffraction,Fourier-transform infrared spectroscopy,vibrating sample magnetometry,and thermogravimetric analysis.The carriers had a regular spherical shape with a mean diameter of 12 nm.The saturation magnetization was 29.25 emu/g for the carriers and 44.49 emu/g for the immobilized agarase,which indicated that agarase had been successfully immobilized onto the supports.The optimal immobilization conditions for 20 mg of the carriers included 4%?v/v?glutaraldehyde,a cross-linking time of 2 h,an immobilization time of 1 h,an immobilization temperature of 4°C,and 850 U of agarase.The properties of immobilized agarase were compared with those of free agarase to assess the feasibility of utilizing the immobilized enzyme for oligosaccharide preparation.The immobilized agarase showed maximal catalytic activity at pH 7.0 and 40°C.After immobilization,the agarase exhibited better thermal and pH stability,as well as good operation stability.The immobilized agarase maintained approximately 40%of its initial activity even after being reused seven times.The K_m?Michaelis-Menten constant?values for immobilized and free agarase were 6.9 and 11.3 mg/mL,respectively.