Immobilisation Of Nitrile Hydratase On Novel Silica And Its Application

Nitrile hydratase catalyzes the hydration of nitriles to their corresponding amides and has important application in the industrial production of acrylamide, nicotinamide and other compounds. Because of the high specificity of NHase, the process of amides production is low energy consumption and less by-products. However, free NHase suffers from low operational stability and difficulty to recovery. To overcome these drawbacks, immobilization of NHase on a suitable support is a promising strategy. Silica is one of the most common supports for immobilized enzyme. It has a wide of sources, a variety of forms and good biocompatibility. So silica is considered to be an ideal carrier for enzyme immobilization. In this study, NHase ES-NHT-118 was chosen as model enzyme, and silica materials were chosen as supports, using encapsulation and cross-linking methods for NHase immobilization. The prepration conditions, catalytic properties and stsbilities of immobilized enzyme were studied. And the immobilized enzymes were used for the preparation of nicotinamide. The details in this study were summarized as follows:Firstly, preparation of biomimetic silica immobilized NHase(Encapsulated NHase) and the study of catalytic properties and stabilities. TMOS was chosen as silicon precursor, PDADMA was used as catalyst, the immobilization process was mild and fast. The immobilized enzyme particles are about 100 nm in diameter, encapsulation effiency reached above 98.0%, activity recovery was 26.7%. The thermal stability, pH stability, operational stability and storage stability were studied. Compared with free enzyme, the stability of Encapsulated NHase has been significantly improved. At the same time, Encapsulated NHase showed good reusability.Secondly, preparation, characterization and catalytic properties of NHase-CLEAs in mesoporous silica material(NHase-CLEAs@MOS). A novel mesoporous silica material—mesoporous onion-like silica(MOS) was used as support, NHase was firstly adsorbed in MOS, then cross-linked by dextran aldehyde. As can be seen from the TEM, MOS has clear multilayer structure, and the distance between each layer is about 15 nm. The preparation conditions were as follows: enzyme concentration was 0.0262 mg/mL, the concentration of dextran aldehyde was 1.43 mg/m L and cross-linking time was 2 h. In this condition, the activity recovery reached 48.2%. The thermal stability, pH stability, mechanical stability and storage stability were investigated. Compared with free enzyme and NHase@MOS, NHase-CLEAs@MOS showed better stability. It was found that using MOS as support for the immobilized NHase has higher affinity than that of using SBA-15 as support.Lastly, Encapsulated NHase and NHase-CLEAs@MOS were used for the hydration of 3-cyanopyridine. The optimized reaction conditions were as follows: temperature was 40 oC, pH was 7.0, NHase activity was 4.36 U. The reuse performance of Encapsulated NHase was better than that of NHase-CLEAs@MOS. Through the dynamic analysis, the Km values of Encapsulated NHase and NHase-CLEAs@MOS were 100 mmol/L and 134 mmol/L, respectively.