Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis

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. The mutations 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 67.6°C. Thus, PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification. Biotechnol. Bioeng. 2016;113: 2350-2357. © 2016 Wiley Periodicals, Inc.

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

“… Aim of the study Protein/Source Criteria Number of tested predictions Number of correct predictions Greatest impact Resolution crystal structure Ref. Enzyme stabilization Endoglucanase ( Hypocrea jecorina ) Cut off value < ΔΔG −1.75 kcal mol −1 43 6 Stabilization (ΔT m = 3.2 °C) 1.62 Å [ 65 ] Phosphotriesterase ( Pseudomonas oleovorans ) Cut off value < ΔΔG −0.72 kcal mol −1 52 32 Stabilization (ΔT m = 8.6 °C) 2.25 Å [ 103 ] T1 Lipase ( Geobacillus zalihae ) One mutation site was selected and exchanged against Val, Ile, Met, Phe, Trp compared to wild type 7 1 Stabilization (+ΔT opt- = 10 °C) 1.5 Å [ 93 ] Thermoalkalophilic lipase ( Bacillus thermocatenulatus ) 3 sites preselected and amino acids were exchanged against Phe, Try and Trp. 9 2 Destabilizing variants (ΔT m = −10 °C) 2.0 Å [ 104 ] Haloalkane dehalogenase (WT and one mutant) Sphingomonas paucimobilis Cut off value < ΔΔG −0.84 kcal mol −1 + visual inspection and MD-simulation <150 5 Stabilization (ΔT m = 3 °C) 0.95 Å [ 105 ] Limonene-1,2-epoxide hydrolase ( Rhodococcus erythropolis ) Cut off (ΔΔG < −1.2 kcal mol −1 ) performed additionally further pre-selection 21 6 Stabilization ΔT m = 6 °C 1.2 Å [ 57 ] Cellobiohydrolase ( Hypocrea jecorina ) 43 mutations selected (ΔΔG < −0.75 kcal mol −1 ) 43 …”

“… Algorithm Standard deviation Accuracy range (min.–max.) R-values FoldX 1.0 to 1.78 kcal mol −1 [ 118 , 116 , 95 ] 0.38 to 0.8 [ 128 ] Average accuracy: 0.69 [ 87 , 103 , 127 , 129 ] 0.29 [ 130 ] to 0.73 [ 118 ] BeatMuSiC 1.2 kcal mol −1 [ 77 ] 0.46 [ 77 ] CUPSAT 1.8 kcal mol −1 [ 77 ] 0.5 [ 102 ] 0.3 [ 77 ] I-Mutant 2.0/3.0 1.2 to 1.52 kcal mol −1 [ 77 , 95 ] 0.48 [ 102 ] to 0.75 [ 127 ] 0.16 [ 77 ] to 0.51 [ 95 ] PoPMuSiC 1.1 kcal mol −1 to 1.32 [ 77 , 95 ] 0.62 [ 129 ] to 0.85 [ 129 ] 0.51 to 0.55 [ 95 , 77 ] mCSM 3.2 kcal mol −1 [ 77 ] 0.23 [ 77 ] ENCoM 1.5 kcal mol −1 [ 77 ] 0.04 [ 77 ] Rosetta-ddG 2.3 kcal mol −1 [ 95 …”

Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

“…Previous study demonstrated that the hydrophobic PPO blocks of Pluronic F127 can interact with hydrophobic amino acids such as Phenylalanine (Phe) on the surface of OPH, and physically associate with OPH to form a noncovalent binding enzyme-polymer complex (Kim et al 2014b). PoOPH M9 (PDB: 4O98) is an engineered OPH belonging to the superfamily of phosphotriesterase (Luo et al 2016), and 10 Phe residues (22 in total) among other hydrophobic residues are located on the surface (Additional file 1: Figure S1). Thus, we modified the process of preparing CLEA and added Pluronic F127 into the reaction mixture after protein precipitation for enzyme-polymer conjugation (Fig.…”

“…In the previous study, we engineered a newly discovered phosphotriesterase (PoOPH M9 , Luo et al 2016) with improved catalytic activity and thermostability for efficient malathion degradation, and demonstrated its effectiveness in removing malathion using free PoOPH M9 in the presence of various detergents (Bai et al 2017), which prompted us to further explore the possibility of preparing immobilized enzymes for potential industrial applications. Herein, we reported a simple method to prepare cross-linked enzyme-polymer conjugates (CLEPC) by directly incorporating Pluronic F127 in the process of carrier-free PoOPH M9 immobilization.…”

High-Throughput, Lysis-free Screening for Sulfatase Activity Using Escherichia coli Autodisplay in Microdroplets

“…5,6 Directed evolution has been used successfully to improve several enzyme properties such as enantioselectivity, [7][8][9] operational stability, [10][11][12] and catalytic efficiency. 13,14 Effective enzyme engineering by directed evolution requires fast, sensitive and reliable screening of large libraries of enzyme variants in order to select the very few improved variants present in these libraries. Current methods for large-scale library testing such as colorimetric colony screening 8,15 or growth selection-based systems 16,17 suffer from narrow dynamic ranges, i.e.…”