Study On The Preparation Of Alginate Oligosaccharides By Immobilized Alginate Lyase

The by-products of kelp which contain plenty of fucodian could be an excellent material for the preparation of alginate oligosaccharides.Alginate oligosaccharides have high water solubility,strong biological activity,easy absorption,anti-tumor,anti-oxidation,cholesterol reduction,immune regulation and other physiological activities,which can be widely used in food,medicine and other fields.However,the existing enzymatic methods of preparing the alginate oligosaccharides have many disadvantages such as the defeats of enzyme sensitivity to environmental conditions,difficulty in recycling and reusing,which makes the exploring a suitable method of preparing alginate oligosaccharides particularly important.Immobilized enzyme showed good application prospects in preparing the alginate oligosaccharides because it was more stable and could be recycled and reused.In this study,magnetic chitosan microspheres were designed and synthesized,and a method of adsorption-crosslinking immobilization of alginate lysase based on magnetic chitosan microspheres was established.And the preparation of alginate oligosaccharides from alginate polysaccharides catalyzed by immobilized alginate oligosaccharides using by-products of kelp as the raw materials was established.The main results are as follows:（1）The preparation and characterization of magnetic chitosan microspheres.The scanning electron microscope（SEM）,fourier transform infrared spectrometer（FTIR）,X-ray diffractometer（XRD）,thermogravimetric analyzer（TG）,physical property measurement system（PPMS）were employed to characterize the morphology,structure,thermal stability and magnetic responsiveness of the magnetic chitosan microspheres which were prepared by the methods of reverse suspension crosslinking using glutaraldehyde as crosslinking agent.The results showed that the magnetic chitosan microspheres which were spherical in shape and micron-sized in particle size had walnut-like folds on the surface.The content of magnetic Fe₃O₄ nanoparticles which were embedded inside the chitosan was about 26.39%in magnetic chitosan microspheres.And the saturation magnetization intensity of magnetic chitosan microspheres was 11.34 emu/g,which indicated that the magnetic chitosan microspheres could be easily separated under the magnetic field.Therefore,magnetic chitosan microspheres were a good immobilization carrier which could recycle the enzymes rapidly under the magnetic field.（2）The preparing and enzymatic property characterization of immobilized alginate lyase.The immobilized alginate lyase was prepared by the methods of adsorption-crosslinking using magnetic chitosan microspheres as the carrier.The effects of the amount of magnetic chitosan microspheres,adsorption time,glutaraldehyde concentration and immobilization time on the rate of enzyme activity recovery were investigated and the structure and enzymatic property of immobilized alginate lyase were also characterized.The results showed that the best rate of enzyme activity recovery of immobilized alginate lyase with 55%was obtained under the following conditions:0.15 g of magnetic chitosan microspheres,2 h of adsorption time,0.2%of glutaraldehyde concentration and 2 h of immobilization time.The alginate lyase could be successfully immobilized on the magnetic chitosan microspheres and had a certain magnetic response which could be separatde rapidly under magnetic field by the anlysis results of FTIR and PPMS.Compared with the free alginate lyase,the optimum temperature of immobilized alginate lyase（35℃）was unchanged while the optimum p H of immobilized alginate lyase（8.0）migrated to an alkaline environment and its tolerance to p H and high temperature was also improved.And the K_mvalues of immobilized alginate lyase was also improved which indicated that the affinity between the immobilized alginate lyase and substrate decreased.The immobilized alginate lyase showed higher operational and storage stability which could retain 51%and 40%of initial enzyme activity after being used for 8 times and stored for 56 days,respectively.These results indicated that immobilized alginate lyase could enconomise cost and achieve continuous production in industrial production due to its higher thermal stability,p H stability,operational stability and easy separation from products.（3）The preparation of alginate oligosaccharides by immobilized alginate lyase.The composition of by-products of kelp was analysised and the preparation of alginate oligosaccharides（enzymatic temperature,enzymatic p H,the amount of enzyme,the concentration of substrate,enzymatic time）were optimized and immobilized alginate lyase after being used many times on the alginate oligosaccharides were investigated.Then the element composition of enzymolysis solution and structure of alginate oligosaccharides prepared by immobilized enzyme were analysised.The results showed the carbohydrate which achieved about 53%was the highest in the composition of by-products of kelp indicating that by-products of kelp were an excellent material for prapartion of alginate oligosaccharides.The highest alginate oligosaccharides with 4.88 mg/m L was obtained under the following conditions:30℃of enzymatic temperature,7.0 of enzymatic p H,0.3%of free alginate lyase,2.5%of substrate concentration and 5 h of enzymatic time by using free alginate lyase.The highest alginate oligosacchides with 4.83 mg/m Lwas obtained under the following conditions:35℃of enzymatic temperature,7.0 of enzymatic p H,0.3 g of immobilized alginate lyase,2%of substrate concentration and 4 h of enzymatic time and the alginate oligosacchaides could retain 46%of the intial by using the immobilized alginate lyase after 5 cycles.The elements of K,Ca,Zn and Mg were high in enzymatic hydrolysates.FTIR showed that the product was alginate oligosaccharides.