Glutaryl 7-amino cephalosporanic acid acylase (glutaryl 7-ACA acylase) (EC 3.5.1.93) is an enzyme commonly applied in bioconversion of glutaryl 7-ACA to 7-ACA. The bacterial strain Hg32 with high and stable activity of intracellular glutaryl 7-ACA acylase was isolated from soil samples in Ha Giang province and identified as one of the Cifrobacfer freundii species (deposited onto GenBank (NCBI) under the accession number H0677190. Based on the reference medium M77, the cultivation conditions of strain C. freundii Hg32 were studied with respect to medium composition and concentrations as well as the mode of glutaric acid addition to induce glutaryl 7-ACA acylase biosynthesis of strain Cifrobacfer freundii Hg32. Resulting in the cellection culture medium of following composition (g/l): yeast extract, 5.0; mono sodium glutamate, 9.0; corn steep liquid, 7.0; casein hydrolysate, 12.0 and inducer glutaric acid, 0.5. Glutaryl 7-ACA acylase by strain Cifrobacfer freundii Hg32 was remarkably influenced by the medium composition and the cultivation conditions. The culture of Hg32 in resulted medium using 3.1 Bioflo 110 fermenter showed that the highest glutaryl 7-ACA acylase activity of intracellular was 476.34 U/l after 27 hours in the culture fermentation. The optimal temperature and pH for this enzyme were 37°C and 8.0, respectively. Metal ions, such as Zn2;, Na;, Mn2+, Fe2+ and [(NH4hFe]+ at the concentrations of 1 and 5 mM, enhanced the glutaryl 7-ACA acylase activity to 10-40 percent. The inhibition of 20-60 percent activity was observed with the presence of Ca2+, Al3+, K+, but the worst inhibitor was Cu2+ which completely inactivated glutaryl 7-ACA acylase.