Malathion is one of the most widely used organophosphorus pesticides in the United States and developing countries. Herein, we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering thermostability. In the first step, iterative saturation mutagenesis at residues lining the binding pocket (CASTing) was employed to optimize the enzyme active site for substrate binding and activity. Hot spots for enhancing activity were then discovered through epPCR-based random mutagenesis, and these beneficial mutations were then recombined by DNA shuffling. Finally, guided by in silico energy calculations (FoldX), thermostability of the variant was improved. Themutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening < 9,000 mutants, the best variant PoOPHM9 showed 25-fold higher activity than wild-type PoOPHM2, with a thermostability (T-50(15)) of 67.6 degrees C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. (C) 2016 Wiley Periodicals, Inc.