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Free keywords:
biocatalysis; directed evolution; DNA shuffling;
malathion; phosphotriesterase
Abstract:
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 (T50
15) of 67.6C.
Thus, PoOPHM9 appears to be an efficient and robust candidate for
malathion detoxification.
