Free Penicillin G Acylase Research Articles

Design of large-pore mesoporous materials for immobilization of penicillin G acylase biocatalyst

In this study, functionalization of large-pore nanoporous silica materials was carried out by condensation of tetraethylorthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES), 3-mercaptopropyltrimethoxysilane (MPTMS), phenyltrimethoxysilane (PTMS), vinyltriethoxysilane (VTES), and 4-(triethoxysilyl)butyronitrile (TSBN), respectively, in the presence of non-ionic surfactant under acidic conditions. The TSBN functionality was subsequently converted to carboxyl group while APTES was further functionalized with glutardialdehyde, a cross linker. The various functionalized materials were used as supports for immobilization of enzyme penicillin G acylase (PGA). Experimental data showed that the functionalized materials except for the material functionalized with MPTMS possess a faster loading kinetics and a higher loading amount of enzyme PGA than the pure-silica counterpart. The enzymatic catalytic activities of the immobilized biocatalysts varied from 52.2 to 167.5 U/g of solid. The glutardialdehyde-activated material displayed the highest initial immobilized enzyme activity and the most stable activity among all the support materials. PGA immobilized on VTES-functionalized nanoporous silica showed the highest initial enzymatic activity (67.7 U/mg of PGA, much higher than that of free PGA (300 U/mg of PGA). Experimental data along with theoretical analysis results indicate that glutardialdehyde is a good cross linker, offering covalent binding of PGA with the support materials while VTES-functionalized nanoporous silica is a very good potential support for physical entrapment of PGA enzyme.

Enzymatic synthesis of β-lactam antibiotics using penicillin-G acylase in frozen media

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various β-lactam antibiotics in ice at −20°C with higher yields than obtained in solution at 20°C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20°C to 25 at −20°C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.