Immobilization Of Fructosyltransferase And D-mannose Isomerase

Fructooligosaccharides and D-mannose,two typical representative of functional carbohydrate,can proliferate bifidobacteria effectively and improve the intestinal environment and their annual outputs exceed 10,000 tons.The technology of immobilized enzyme has the greatest potential to commercial production owing to its many advantages such as high enzyme utilization rate and low production cost.In this study,Aspergillus niger AS0023 and Bacillus subtilis SK48.001 were fermented to obtain crude enzyme powder,then immobilized fructosyltransferase and D-mannose isomerase with different carriers,respectively.Optimum conditions and enzymatic properties were also explored in order to provide a theoretical basis for industrial applications.With ten kinds of ion exchange resin and macroporous adsorption resin as carriers and glutaraldehyde as cross-linking agent,immobilized fructosyltransferase was studied by adsorption and cross-linking.The results showed that the most effective one was Duolite A568which is a highly porous granular weak base anion exchange resin.When added 285 U/g resin,carried out for 7-hour adsorption at 24°C and pH 5.0,then adjusted the volume concentration of glutaraldehyde to 0.01%and crosslinked at 4°C for 2 h,the recovery rate reached 155.19U/g resin,which came to the highest and was 1.98 times higher than that before optimization.The optimum temperature of the immobilized enzyme was 60°C,which was higher than the free enzyme.It achieved maximum activity at pH 5.5,while the free enzyme was 5.0.And the Michaelis constant was 930.94 mmol/L,while the free enzyme was 587.75 mmol/L,indicating the former had a lower affinity for substrates.After 8 batches of reaction,the enzyme retained more than 45%of its initial activity,which meant Duolite A568 had good immobilization performance.D-mannose isomerase was immobilized on the same types of resin,in which Amberlite FPA90 Cl performed the best.The optimum conditions for the immobilization were as follows:enzyme load of 26 U/g resin,4-hour absorption at 28°C and pH 8.5,cooled at 4°C for 30 min,then added the glutaraldehyde of which concentration was 0.04%and 2-hour cross-linking at4°C.Finally,the recovery yield of the immobilized enzyme reached over 70%and the enzyme activity was 19.21 U/g resin,which was 1.11 times higher than that before optimization.The optimum temperature and pH were 45°C and 8.0,respectively.Besides,the temperature and pH stability were both better than the free enzyme.It also presented higher K_m than the free enzyme and retained 80.8%of its initial enzyme activity after reuse for eight times,showing that the immobilized enzyme had good operational stability.In this study,coprecipitation was employed to synthesize magnetic Fe₃O₄ nanoparticles,then selected chitosan as the modifier to prepare chitosan-coated magnetic Fe₃O₄ nanoparticles in order to enhance its biocompatibility.Then immobilized D-mannose isomerase by product above,and determined the optimal immobilization conditions by single factor method:the amount of enzyme was 30 U/g carrier,the immobilization pH was 6.5,and the temperature was fixed at 20°C for 2 h.After some experiments on the enzymatic properties of the immobilized enzyme,the results showed that the optimum reaction temperature was 50°C,and the optimum pH was 7.5.The enzyme activity retained 26.19%after repeated 6 cycles,which indicated that it was feasible to immobilize D-mannose isomerase with CS-MNPs.This material is convenient for controlling the initiation and termination of the reaction,and is of great significance for the rapid separation of enzymes and reaction mixtures in actual production,laying a foundation for subsequent research.