Preparation And Catalytic Properties Of Penicillin G Acylase Immobilized On Macroporous Material

As one of the most important industrial biocatalysts, penicillin G acylase (PGA, EC3.5.1.11) has been used widely in the production of6-aminopenicillanic acid and thesemi-synthetic antibiotics. As one of the biocatalysts, PGA has excellent properties of highselectivity, specificity and activity. Furthermore, reaction conditions are moderate. However,low operational stability and the difficulty to recovery limit the wide use of PGA at theindustrial scale. Hence the idea of immobilizing PGA on prefabricated carriers appears as agood strategy to surmount these obstacles. Macroporous material, with wide channels and agood permeability, can minimize the diffusion resistance and then significant enhancementin diffusivities of guest species and active site accessibility can be observed. Thus themacroporous material would be rewarding to study more to immobilize enzyme. In thispaper, macroporous material, as a supporter, is used for immobilizing PGA by covalentbond. The details in this study were summarized as follows:(1) The hierarchically macro-mesoporous monolith was prepared using high internalphase emulsions (HIPEs) as templates and modified with amino and epoxy groups. Theproperties of the immobilized PGA were studied. Additionally, the stability as well as thekinetic properties was also evaluated. Compared with free PGA, the immobilized PGA hadwider proper temperature and pH range.(2) Inspired by the role of catecholamines in mussel adhesion, norepinephrine canmodify the surface of the carrier via oxidative polymerization. The poly(norepinephrine)coating is expected to retain residual quinine groups that are reactive toward amine andthiol contained in protein. Thus it is a facile approach to immobilize enzyme utilizingnorepinephrine by covalent linkage. Norepinephrine was utilized to modify the silica andthe functionalized silica was used to immobilize PGA. The immobilized conditions wereoptimized and the stability of immobilized PGA was investigated. Compared with freePGA, the immobilized PGA exhibited better stability. The kinetic parameters were alsostudied. In order to verify the possibility to apply in practical, the immobilized PGA wasapplied in a packed bed bioreactor and used to hydrolyze Pen G. The immobilized PGA hadhigh activity after15d continuous hydrolysis presenting possibility to apply in practical.(3) In order to investigate the possibility to modify hydrophobic material by norepinephrine, polystyrene was used as a model to be modified and used for immobilizingPGA. The immobilizing process was optimized. The stability and kinetic parameters werealso investigated. The prepared immobilized PGA was padded in chromatographic columnto hydrolyze Pen G. It was found that norepinephrine can modify polystyrene and theimmobilized PGA has excellent stability.