Peng Wang1,2,3, Chen Shen4,5, Qinqin Cong4, Kaili Xu4, Jialin Lu4. 1. College of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang, 050018, China. pwang@hebust.edu.cn. 2. State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science and Technology, Shijiazhuang, 050018, China. pwang@hebust.edu.cn. 3. Hebei Province Pharmaceutical Chemical Engineering Technology Research Center, Shijiazhuang, 050018, China. pwang@hebust.edu.cn. 4. College of Chemical and Pharmaceutical Engineering, Hebei University of Science and Technology, Shijiazhuang, 050018, China. 5. State Key Laboratory Breeding Base-Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science and Technology, Shijiazhuang, 050018, China.
Abstract
BACKGROUND: Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. RESULTS: In this study, the beta-lactamase from Ochrobactrum tritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrum tritici, the β-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C β-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrum tritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 ℃ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 μM and a kcat value of 25.28 s-1 respectively. CONCLUSIONS: OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.
BACKGROUND: Biodegradation of antibiotics is a promising method for the large-scale removal of antibiotic residues in the environment. However, the enzyme that is involved in the biodegradation process is the key information to be revealed. RESULTS: In this study, the beta-lactamase from Ochrobactrum tritici that mediates the biodegradation of penicillin V was identified and characterized. When searching the proteins of Ochrobactrum tritici, the β-lactamase (OtLac) was identified. OtLac consists of 347 amino acids, and predicted isoelectric point is 7.0. It is a class C β-lactamase according to BLAST analysis. The coding gene of OtLac was amplified from the genomic DNA of Ochrobactrum tritici. The OtLac was overexpressed in E. coli BL21 (DE3) and purified with Ni2+ column affinity chromatography. The biodegradation ability of penicillin V by OtLac was identified in an in vitro study and analyzed by HPLC. The optimal temperature for OtLac is 32 ℃ and the optimal pH is 7.0. Steady-state kinetics showed that OtLac was highly active against penicillin V with a Km value of 17.86 μM and a kcat value of 25.28 s-1 respectively. CONCLUSIONS: OtLac demonstrated biodegradation activity towards penicillin V potassium, indicating that OtLac is expected to degrade penicillin V in the future.
Authors: H W Leung; T B Minh; M B Murphy; James C W Lam; M K So; Michael Martin; Paul K S Lam; B J Richardson Journal: Environ Int Date: 2011-03-29 Impact factor: 9.621