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Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis
Author(s) -
Luo XiaoJing,
Zhao Jian,
Li ChunXiu,
Bai YunPeng,
Reetz Manfred T.,
Yu HuiLei,
Xu JianHe
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26012
Subject(s) - thermostability , mutagenesis , in silico , malathion , dna shuffling , chemistry , directed evolution , mutant , protein engineering , saturated mutagenesis , enzyme , biochemistry , biology , pesticide , gene , agronomy
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 Po OPH M2 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. The mutations extend from the core region to the enzyme surface during the evolutionary pathway. After screening <9,000 mutants, the best variant Po OPH M9 showed 25‐fold higher activity than wild‐type Po OPH M2 , with a thermostability ( T 50 15 ) of 67.6°C. Thus, Po OPH M9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350–2357. © 2016 Wiley Periodicals, Inc.