Immobilization Of Phenylalanine Ammonia Lyase Based On Biomimetic Silicification And Its Catalytic Properties

Compared to existing immobilization routes, the advantages of biomimetic silicas technology for immobilized enzymes include their mild processing conditions, simple bench-top one-step procedure, low cost and short preparation time, in addition to excellent immobilization efficiencies, high activity and improved stability. Successful preparation of biomimetic silicification has been used from a broad range of enzymes. Cross-linked enzyme aggregates technology (CLEAs) has been regarded a promising free-carrier immobilization. The advantages of CLEAs include simple procedure and low cost, as well as increased stability. However, correspond with biomimetic silica; CLEAs may still be considered too soft, serious diffusion problems and difficulty of recovery for many industrial applications. The drawbacks of CLEAs restrict its industrial applications. To solve the issues of immobilization technology mentioned above, we prepared respectively the encapsulated CLEAs in biomimetic silica and mesoporous CLEAs-silica composite microparticles by the combination of biomimetic silicas technology and CLEAs. For comparison, the conventional CLEAs, the encapsulated PAL in biomimetic silica and the encapsulated CLEAs in biomimetic silica were also prepared. Furthermore, catalytic properties of all immobilized enzymes were investigated.1) Preparation and enzymatic characteristics of the encapsulated PAL in biomimetic silica. The preparing conditions of the encapsulated PAL in biomimetic silica were optimized by single factor experiment. These factors include polyethyleneimine (PEI) concentration, tetramethoxysilane (TMOS) concentration, and buffer, pH, as well as PAL concentration. Response surface methodology was used to optimize the preparing conditions. Furthermore, the characteristics of immobilized PAL were also investigated. The results showed that the optimal conditions of the encapsulated PAL in biomimetic silica were TMOS concentration at 1%(w/v), PEI concentration at 2.4 mg/ml, and 1 mL PAL(2.85×10-6U). Under optimized conditions, the maximal activity recovery of the encapsulated PAL in biomimetic silica was respectively 28%. The Scanning Electron Microscope (SEM) showed that the immobilized PAL had irregular particles with a size of 50-200nm. The optimal pH and temperature of the encapsulated PAL in biomimetic silica was 50℃ and 8.5, respectively. Compare to free PAL, stability of the encapsulated PAL in biomimetic silica against high temperature, pH and denaturant were significantly enhanced.2) Preparation and enzymatic characteristics of the encapsulated CLEAs in biomimetic silica (CLEAs-Si).In order to improve mechanical strength of CLEAs, CLEAs were entrapped in biomimetic silica by the combination of biomimetic silicas technology and CLEAs. Transmission Electron Microscopy (TEM) and fluorescence microscope demonstrated that CLEAs have been successfully encapsulated in the biomimetic silica. SEM showed that CLEAs-Si particles had regular spherical shape with a size of 50-500 nm and a relatively lower polydispersity. The optimal pH and temperature of CLEAs-Si was 50 ℃and 8.5, respectively. Furthermore, Compare to conventional CLEAs and biomimetic silica, stability of CLEAs-Si against high temperature, pH and denaturant were significantly increased, and CLEAs-Si exhibited excellent reusability.3) Preparation and enzymatic characteristics of mesoporous CLEAs-silica composite microparticles (P-CLEAs-Si).In order to decrease diffusion limitations, the gelatinized starch was selected as the pore-making agent to decrease diffusion limitations. The porosity of P-CLEAs-Si particles was prepared by encapsulated simultaneously CLEAs and starch within biomimetic silica, and subsequently a-amylase was added to remove gelatinized starch in CLEAs and biomimetic silica. SEM and TEM showed that P-CLEAs-Si particles had regular spherical shape with mesopores and a relatively rough surface. However, these phenomenons were not observed in CLEAs-Si. Furthermore, the highest PAL activity in P-CLEAs-Si was displayed at 2 g/L starch concentration. The optimal pH and temperature of P-CLEAs-Si was 50 ℃and 8.5, respectively. Furthermore, Compare to conventional CLEAs and biomimetic silica, stability of P-CLEAs-Si against high temperature, pH and denaturant were significantly increased, and CLEAs-Si exhibited excellent reusability.