| While conventional methodologies of chemical processes have been developed in the past decades to a level allowing production, separation and analytical determination of an enormous range of sophisticated products, alternative methodologies that are not only efficient and safe but also environmentally benign and resource-and energy-saving, are being increasingly sought. One of the most promising strategies to achieve these goals is the utilization of enzymes. In addition to the unquestionable advantages, there exists a number of practical problems in the use of enzymes. To these belong:the instability of their structures once they are isolated from their natural environments, and their sensitivity both to process conditions. Also, most enzymes operate dissolved in water in homogeneous catalysis systems, which contaminates the product and as a rule cannot be recovered in the active form reaction mixtures for reuse. To overcome these limitations, enzyme immobilization is one of the most successful methodologies. With the widespread application of immobilized enzymes in synthetic chemistry and industry, the immobilization of enzymes on solid supports is an area of intense research. As conventional supports for enzymatic immobilization, such as polymeric organic materials and inartificial macro-molecules, inorganic materials possess large specific surface area and unique structure and have drawn growing interest of researchers. Among inorganic materials, silica materials for the immobilization of enzymes have been extensively because they are environmentally more acceptable, structurally more stable and chemically more resistant to organic solvents and microbial attacks. In this study, hollow silica nanotubes synthesized via s sol-gel route using nano-sized needle-like calcium carbonate inorganic templates were employed as a support for immobilization of enzymes, such as penicillin G acylase, glucose oxidase and lysozyme. The main contents and finding are summarized as follows.1、Producing needle-like calcium carbonate by carbonation carried out in rotating packed bed (RPB) reactor were investigated in this paper. Under the appropriate conditions, well-dispersed needle-like calcium carbonate with average diameter of0.2～0.5μm, average length of3.0～5.0μm and88.75%aragonite phase could be yielded successfully.2、To acquire better material structure and performance, we explored a novel method of needle-like calcium carbonate as a inorganic template to synthesize hollow silica nanotubes. Introducing CTAB as a pore channel directing agent, using TEOS as organic silica source and utilizing orthogonalexperiments, hollow silica nanotubes with good morphology were successfully obtained, the conditions of the optimal parameter combination were shown as follow:the reaction temperature (15℃); SiO2weight percentage (20%); CTAB weight percentage (50%). silica nanotubes with pore sizes of about6.5nm, a shell thickness of30-40nm and surface area of749m2/g have been prepared under optimal synthesis conditions.3、Hollow silica nanotubes synthesized via a sol-gel route using nano-sized needle-like calcium carbonate as a inorganic template were employed as a support for immobilization of penicillin G acylase biocatalyst. Effect of various factors, such as loading temperature and ratio of carriers to free penicillin G acylase, on the catalytic activity of the immobilized penicillin G acylase was investigated. The results show that under optimized conditions the relative loading amount and the total activity yield of immobilized enzyme amounts to97.20%and88.80%, respectively. Several advantages, ie. the rapid immobilization of penicillin G acylase onto hollow silica nanotubes, the high tolerability to the PH, the less sensitivity to the temperature and the improved storage stability render hollow silica nanotubes potential support materials for enzyme immobilization. Through the study of kinetics of penicillin G potassium hydrolysis by free and immobilized penicillin G acylase, the Michaelis-Menton constant(Km) and the most reacting velocity(V) of free penicillin G acylase are3.23×10-2mol/L and9.3×10-3mol/(L· min), respectively. For those of immobilized penicillin G acylase are6.7X10-2mol/L and3.7×10-3mol/(L· min), which indicated that the appetency between enzymatic molecules and substrates would appear a descending trend after immobilization of penicillin G acylase on hollow silica nanotubes 4、Three different morphology silica materials i.e. hollow silica nanotubes, hollow silica nanospheres and solid silica nanoparticles were utilized for immobilization of lysozyme. The comparative study of three silica materials for the immobilization of lysozyme indicated that the amount of immobilized lysozyme on solid silica nanoparticles was186mg/g silica, and while that of immobilized lysozyme within hollow silica nanotubes and hollow silica nanospheres could, respectively, reach up to351mg/g silica and385mg/g silica. Among the three kinds of silica supports, it was obvious that the hollow silica nanospheres represented the highest immobilization ability, whereas the specific activity of immobilized lysozyme on silica nanospheres (1.38×106unit/g) was slightly lower than that of immobilized lysozyme on silica nanotubes (1.46×106unit/g). Meanwhile, we also found that the adsorption process of lysozyme onto hollow silica materials could be divided into two stages. Good hollow structure and large pore size of silica materials would enhance the amount of immobilized large molecules, such as lysozyme, within silica materials. An interesting phenomenon is that the relatively airtight structure of hollow silica nanospheres has double effects on enzymatic immobilization. On one hand, the relatively airtight structure of hollow silica nanospheres is more useful for the molecules of lysozyme to be captured and restricted into the void space of silica nanospheres. Thus, this facilitates to enhance the amount of immobilized enzyme onto hollow silica nanospheres. But on the other hand, the relatively airtight structure might affect the diffusion/mass-transfer processes upon substrates entering in and products flowing out through the shell of hollow silica nanoshpheres, leading to the decrease of the immobilized lysozyme activity. The comparative study of immobilization of lysozyme onto the three different silica materials indicated clearly that hollow silica nanomaterials would be more potential candidates for enzyme immobilization than solid silica nanoparticles.5、The prepared hollow silica nanotubes were utilized for glucose oxidase immobilization. An interesting phenomenon is that the enzyme activity first increases and then decreases with further increasing surface coverage of the carriers in this section, suggesting that there exists an optimum amount of bound protein suitable for the most efficient expression of enzyme activity. The intact secondary structure of the protein in the adsorbed glucose molecules indicates that hollow silica nanotubes are stable and compatible with the bioenvironment. |
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