Abstract
Medicinally useful cephalosporins are produced by fermentation products with further modifications. Although cephalosporins are superior antibiotics compared with penicillins, their distribution is limited because the processes for further chemical modifications are complicated and costly. DACS, a nonheme-iron(II) and 2-oxoglutarate-dependent oxygenase, catalyzes the 3’-hydroxylation of DAOC in the biosynthesis of cephalosporins. It can convert its natural substrate analogue DAOG to DAG with relatively low yield. The goal of this study is to raise the accessibility of DACS to DAOG via protein engineering techniques. We began to design mutations via site-directed mutagenesis at the residues near the binding pocket or C terminal, including F268A, R270L, A185Y, V279I, V307A, T308A, T308K, M309L and H310L. Only T308A showed slightly higher activity. Therefore, we applied random mutagenesis such as chemical mutagenesis, site-directed random mutagenesis, and error-prone PCR. After screening 6600 transformants by several steps, we obtained 69 mutants and sequenced them. The three mutants S218D, A80G and A57V showed the highest activities. Furthermore, the A57 was mutated to K, I, E, L and G. Kinetic parameters of the recombinant DACSs were measured by assays under the optimized condition. The enzyme product of the A57E mutant showed the highest activity (kcat/KM = 2.59±0.12/Msec) which was about 1.86 fold of that of the wild type (kcat/KM = 1.39±0.29/Msec). The data showed that the well-developed screening methods could successfully help us to find the desired mutants derived from random mutagenesis and further manipulations at those residues may improve enzymatic activities.