Immobilisation Of Nitrile Hydratase On Magnetic Mesoporous Silica And Its Application

Nitrile hydratase?NHase?is an important industrial enzyme that can efficiently catalyze the hydration of nitriles to produce corresponding amides.The process of amides production is low energy consumption and less by-products,and it fit in with requirement of economic and environmental friendly green chemical industry.However,the free NHase has poor stabilities,it's difficult to separate free NHase from the reaction mixture,and free NHase lose activity easily under the influence of the surrounding environment.To overcome these drawbacks,immobilization of enzyme on a suitable support is a promising strategy.Magnetic mesoporous silica material which combines the advantages of mesoporous structure and magnetic properties is an ideal support for enzyme immobilization owing to its high specific surface area,tunable pore size,large pore volume,good biocompatibility,high chemical stability,easy surface modification and easy separation.In this study,magnetic mesoporous silica material was chosen as support,using adsorbing,cross-linking and covalent binding methods for immobilization of NHase.The prepration conditions,catalytic properties and stabilities of immobilized enzyme were studied,and the immobilized enzyme were used for the production of nicotinamide and cephalexin.The details in this study were summarized as follows:Firstly,mesoporous silica nanoparticles?TA-MSNs?were synthesized using tannic acid as templating agents,NHase was immobilized on TA-MSNs through the carrier-bound CLEAs method to prepare immobilized NHase?CLNHAs@TA-MSNs?.TA-MSNs was monodisperse spherical particles with a diameter of around 200 nm.Its specific surface area was 438.5 m²/g,and the diameter and cumulative volume of the pores were 9.559 nm and1.245 cm³/g,respectively.The optimum temperature of CLNHAs@TA-MSNs was 40^?C,and the optimum pH of CLNHAs@TA-MSNs was 7.0.Compared with free NHase,CLNHAs@TA-MSNs exhibited improved thermal,pH,mechanical and storage stability.Furthermore,CLNHAs@TA-MSNs was applied in production of nicotinamide,the tolerance of CLNHAs@TA-MSNs to high concentration of substrate was better than that of free NHase,and yield of nicotinamide could still reach 29.31%after six cycles of reaction.K_m value of CLNHAs@TA-MSNs was larger than that of free NHase,indicating lower affinity of substrate for CLNHAs@TA-MSNs compared to free NHase.V_max,K_cat and Kcat/Km values of CLNHAs@TA-MSNs was smaller than that of free NHase,indicating the catalytic efficiency of CLNHAs@TA-MSNs decreased in comparison with free NHase.Secondly,magnetic mesoporous silica nanoparticles?TA-MMSNs?were synthesized using tannic acid as templating agents and Fe3O4 as magnetic source,NHase was immobilized on TA-MMSNs through the carrier-bound CLEAs method to prepare immobilized NHase?CLNHAs@TA-MMSNs?.TA-MMSNs was monodisperse spherical particles with a diameter of around 250 nm and a magnetization saturation value of 35.26emu/g.Its specific surface area was 423.4 m²/g,and the diameter and cumulative volume of the pores were 9.349 nm and 1.071 cm³/g,respectively.The optimum temperature of CLNHAs@TA-MMSNs was 40^?C,and the optimum pH of CLNHAs@TA-MMSNs was7.0.Compared with free NHase,CLNHAs@TA-MMSNs exhibited improved thermal,pH,mechanical and storage stability,and tolerability of trypsin.Furthermore,CLNHAs@TA-MMSNs was applied in production of nicotinamide,the tolerance of CLNHAs@TA-MMSNs to high concentration of substrate and organic solvent was better than that of free NHase,and yield of nicotinamide could still reach 29.74%after seven cycles of reaction.K_m value of CLNHAs@TA-MMSNs was larger than that of free NHase,indicating lower affinity of substrate for CLNHAs@TA-MMSNs compared to free NHase.V_max,K_cat and K_cat/K_m values of CLNHAs@TA-MMSNs was smaller than that of free NHase,indicating the catalytic efficiency of CLNHAs@TA-MMSNs decreased in comparison with free NHase.Lastly,immobilized NHase and penicillin G acylase?PGA?was prepared with amino-functionalized TA-MMSNs?TA-MMSNs-NH2?as support using covalent binding method to obtain double enzyme catalytic system?NHase-PGA@TA-MMSNs-NH2?.Specific surface area of TA-MMSNs-NH₂ was 276.5 m²/g,it had a magnetization saturation value of 30.10 emu/g,and its diameter and cumulative volume of the pores were 9.479 nm and 0.4380 cm³/g,respectively.NHase-PGA@TA-MMSNs-NH₂ was applied in production of cephalexin,and the reaction conditions were optimized using response surface methodology.When the reaction temperature was 34.74 ^oC,the reaction pH was 7.75,the reaction time was 6.26 h and the amount of enzyme was 56.76 mg,the yield of cephalexin could reach 85.37%.After four cycles of reaction,the yield of cephalexin could still reach27.19%.