Functionalization Of Mesoporous Materials For Immobilization Of Penicillin G Acylase

Penicillin G acylase (PGA) is a key enzyme used in the pharmaceutical industry for production of β-lactam antibiotics, because it can catalyze the hydrolysis of penicillin G potassium salt to produce6-aminopenicillanic acid (6-APA), which is an important intermediate for synthesis of several semisynthetic β-lactam antibiotics. Unfortunately, free PGA’s low thermal and solvent stability, impossibility of reusability and difficult purification of products inhibit its wide industrial applications. However, these disadvantages can be overcome by immobilization of free enzymes on various solid materials, which provides many distinct advantages including enhanced stability, easy separation from reaction mixtures and then recycling, possible modulation of catalytic performances, effective prevention of enzyme contamination in the products, and easier prevention of microbial growth. Mesostructured cellular foams (MCFs) are composed of uniformly sized, large spherical cells which are interconnected by uniform windows to create a continuous3D pore system. The interconnected nature of large uniform mesopores is favorable for immobilization of enzyme and transportation of substrates and products. Therefore, we synthesized a series of mesoporous materials functionalized with organo-functional groups for PGA immobilization, and investigated the structure of mesoporous materials and the performance of immobilized PGA. The meaningful conclusions are drawn as follows:1. KIT-6and MCFs with continuous3D pore systems were used as efficient supports for immobilization of penicillin G acylase. The results show that PGA/MCFs behave higher adsorptin speed, higher loading amount of PGA and enzymatic activity than PGA/KIT-6, because MCFs with larger pore sizes and pore volume are favorable for immobilizing more enzyme molecules to increase the activity.The operational stabilities of PGA/KIT-6and PGA/MCFs retain only72%and77%of its intial activity after recycled for10times, respectively. The reason is that PGA is immobilized on pure silicas via a relatively weak physical adsorption and PGA can be eluted during the reaction procedure, leading to a continuous decrease in the enzymatic activity of PGA immobilized on pure silicas in the repeated use. 2. The aldehydepropyl-functionalized mesostructured cellular foams (CHO-MCFs) were prepared by post-synthetical functionalization of MCFs with trimethoxysilylpropanal (TMSP), and used as efficient supports for immobilization of PGA. The results show that the aldehydepropyl groups have been grafted successfully on the surface of MCFs, and after functionalization, the BET surface area and pore volume of CHO-MCFs decrease, but the ultra-large and continuous3D mesoporous structure of CHO-MCFs are retained to be beneficial for immobilization of PGA with large size and diffusion of substrates and products, which results in high enzymatic activity. PGA is immobilized covalently on CHO-MCFs via the reaction to produce Schiff’s base between the free amino groups of lysine residues of PGA and the aldehyde groups on the surface of CHO-MCFs, which greatly increases the operational stability of the immobilized PGA with little activity loss due to the short-chain groups of aldehydepropyl grafted on the surface of CHO-MCFs. PGA/CHO-MCFs-10shows the initial enzymatic activity of8895U/g and retains93.0%of its initial activity after recycled for10times.3. The aldehyde-functionalized mesostructured celluar foams (A-MCFs) were successfully prepared by copolymerization of vinyl groups on the surface of vinyl-functionalized mesostructured cellular foams (V-MCFs) with vinyl groups of methacrolein, and used for covalent immobilization of PGA. Effects of functionalization with the organo-functional groups on the physicochemical properties of V-MCFs and A-MCFs, and the enzymatic activity and the operational stability of the immobilized PGA were investigated. A-MCFs-15%sample retains the characteristic ultra-large and continuous3D mesoporous structure of MCFs with high surface area and large pore volume, which is beneficial for immobilization of PGA and diffusion of substrates and products and thus results in high enzymatic activity of the immobilized PGA. PGA is immobilized covalently on A-MCFs sample via the reaction to produce Schiff’s base between the free amino groups of lysine residues of PGA and the aldehyde groups on the surface of A-MCFs, which greatly increases the operational stability of the immobilized PGA. PGA/A-MCFs-15%sample shows the initial enzymatic activity of9531U/g and retains87%of its initial activity after recycled for10times.4. The epoxy-functionalized mesostructured celluar foams (E-MCFs) were successfully prepared by copolymerization of vinyl groups on the surface of vinyl-functionalized mesostructured cellular foams (V-MCFs) with vinyl groups of allyl glycidyl ether (AGE), and used for covalent immobilization of PGA. The physicochemical properties of E-MCFs were characterized, and effects of epoxy contents on the loading amount of PGA, the enzymatic activity and the operational stability of the immobilized PGA were investigated. E-MCFs-10sample retains the characteristic ultra-large and continuous3D mesoporous structure of MCFs with a high BET surface area of559m2/g and a high pore volume of2.0cm3/g, which is beneficial for immobilization of PGA and thus results in high enzymatic activity of the immobilized PGA. PGA is immobilized covalently on E-MCFs sample and thus greatly increases the operational stability of the immobilized PGA. PGA/E-MCFs-10sample shows the initial enzymatic activity of9679U/g and retains90%of its initial activity after recycled for10times, and has better catalytic performance than PGA/CHO-MCFs-10and PGA/A-MCFs-15%.