Substitution of the Catalytic Metal and Protein PEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase Lucia Perezgasga & Lorena Sánchez-Sánchez & Sergio Aguila & Rafael Vazquez-Duhalt Received: 16 October 2011 /Accepted: 14 December 2011 / Published online: 17 January 2012 # Springer Science+Business Media, LLC 2012 Abstract Phosphotriesterase, a pesticide-degrading enzyme, from Flavobacterium sp. was cloned and expressed in Escherichia coli. The catalytic zinc ions were replaced by cobalt atoms increasing the catalytic activity of phosphotriesterase on different pesticides. This metal substitution increased the catalytic activity from 1.4 times to 4 times according to the pesticide. In order to explain this catalytic increase, QM/MM calculations were performed. Accordingly, the HOMO energy of the substrate is closer to the LUMO energy of the cobalt- substituted enzyme. The chemical modification of the enzyme surface with poly(ethylene glycol) increased the thermostability and stability against metal chelating agents of both metal phosphotriesterase preparations. Keywords Phosphotriesterase . Chemical modification . Cobalt substitution . Stability . QM/MM Introduction A diversity of pesticides is widely used for agricultural, domestic, and industrial applica- tions. Together with chemical weapons, pesticides are the only chemicals deliberately made to be toxic and introduced directly into the environment. Only 5% of pesticides reach the target organism. The rest runs off into water or dissipates in the soil or air. Drift from landscape application ranges from 12 feet to 15 miles [1]. Pesticides can be absorbed through the skin, swallowed, or inhaled. More serious effects appear to be produced by direct inhalation of pesticide sprays than by absorption or ingestion of toxins [2]. Adverse health effects are originated by pesticide exposure, such as respiratory disorders, dermal damages, neurological deficit, memory disruption, abortions, and malformations in born infants [3]. Appl Biochem Biotechnol (2012) 166:1236–1247 DOI 10.1007/s12010-011-9510-x L. Perezgasga : L. Sánchez-Sánchez : S. Aguila : R. Vazquez-Duhalt (*) Instituto de Biotecnología, UNAM, Av. Universidad #2001 Col. Chamilpa, C.P. 62250 Cuernavaca, Morelos, Mexico e-mail: vazqduh@ibt.unam.mx