Studies On Synthesize Of Mesoporous Materials And Application In Immobilization Of Lignin Peroxidase

Lignin peroxidase (LiP) has a great capacity for degrading recalcitrant xenobiotics. However, unable to repeatedly use the free enzyme along with its weak stability are two existing obstacles limiting its practical application, prompting the investigation of enzyme immobilization as a possible solution. The present work carried out a series of steps for preparing capable carrier materials and exploring available immobilization methods.Mesoporous silicas with various morphologies and structures were synthesized using Pluronic P123 as template and 1,3,5-triisopropylbenzene(TIPB) as a swelling agent in an acetic/acid sodium buffer solution. The effects of different silica sources, TIPB amount, initial synthesis pH, and temperature were studied. The experimental results were as follows: Samples synthesized using silica sources such as tetramethoxysilane(TMOS), tetraethylorthosilicate(TEOS), and sodium silicate solution(NaSi) produced a "grape", "ping-pong" and "fringe" morphology, respectively. The pore size of mesoporous silica molecular sieves greatly expanded with increasing amounts of TIPB. However, this resulted in poor structural regularity, resulting in its morphology to change from rod-like sub-particles to monoliths. With increasing pH, the sample morphologies experienced a transformation from monoliths to spheres, along with an observed structural transformation from ordered hexagonal meso-structure to vesicles. When the initial synthesis temperature was low, the sample morphologies were regularly particles with a two-dimensional hexagonal structure; at increased temperatures, the particle diameter grew larger and eventually became hollowed spheres. The best synthesized condition was as follows: choosing TMOS as silica source, th amount of TIPB was 1.0g, and the initial synthesis pH and temperature were 3.5 and 12℃The mesoporous materials obtained under the best synthesized condition were utilized as carriers for the immobilization of (LiP). Immobilization time along with the amount of enzymes added were investigated in terms of their effect on the amount of immobilized enzyme synthesized and its activities. Results showed that, as the mass ratio of enzyme and mesoporous material was 76.8mgE/gMS,immobilizing time was 12 h, the largest amount of immobilized enzyme (8.87 mgE/gMS) and the highest apparent activity of immobilized LiP (82.9 U/mg) were achieved. Compared with free LiP, the pH stability and thermal stability of immobilized LiP were significantly improved. Almost 30% of the initial activity was preserved even after six usages.The surface of mesoporous material was modified by the -NH₂ groups via silanization reagents (3-Aminopropyl) triethoxysilane (APTES) and [3-(2-Amino ethyl) aminopropyl] trimethoxysilane (AEAPMDS). The–NH₂ modified samples were used as carriers for the immobilization of LiP. The–NH₂ samples modified by APTES and AEAPMDS exhibited high adsorption capacity (4.63mg/g and 4.49mg/g, respectively), while the apparent activity were 10.22U/mg and 10.55U/mg, respectively.The mesoporous materials containing–NH₂ were activated via glutaraldehyde, 1,4-phenylene diisothiocyanate, cyanotic chloride and water-soluble carbodiimide and then used for immobilization of LiP. The results showed that the best performance of immobilized LiP was obtained by the method using cyanotic chloride. The enzyme loading was 12.15mg/g, and the apparent activity was 541.9U/mg. Immobilized LiP had stronger acid resistance, but more sensitivity towards temperature. There was no apparent change in activity after ten consecutive centrifugations. Furthermore, Acid Orangeâ…¡(0.25mM) was used as the substrate for degradation capacity using immobilized LiP. The percentage of decolorization was 30% after 200s in the reaction system. Almost 60% of the dye could be decolorized after 5 times recycled use.