Organophosphorus Hydrolase:Resource,Structure And Engineering

Organophosphates(OPs)are among the most toxic synthetic compounds and are widely used as agricultural pesticides and nerve agents.Organophosphorus hydrolases(OPHs)degrade the phosphate ester bond of theses compounds,thereby reducing the tocixity,which are reliable reagents for bioremediation and biodetoxification.To date,efficient and stable OPHs with high expression levels are still limited.Besides,Malathion is one of the most widely used organophosphorus pesticides in USA and developing countries for preventing insects on crops,vegetables,tobacco and tea.Enzymes with high hydrolysis rate against malathion were rarely reported.We aimed to obtain new OPHs with high efficiency and robustness by employing genetic engineering and protein engineering techniques.Also,the recombinant enzyme was overexpressed in Pichia pastoris and immobilized enzymes were prepared to improve its application potential in the biodegradation area.Firstly,PoOPH from Pseudomonas oleovorans was discovered from our OPH library containing 42 recombinant homologous enzymes.It exhibited good thermostability and latent phoshotriesterase activity.Surprisingly,PoOPH exhibited a high latonase activity,which was considered to be its native activity.Sequence analysis revealed the conservation of residues His250 and Ile263 and site-directed mutagenesis at these crucial residues enhanced the phosphotriesterase(PTE)activity.The best variant PoOPHM2 carrying H250I/I263W double mutation displayed 6962-and 106-fold improvements in catalytic efficiency for degradation of methyl-parathion and ethyl-paraoxon respectively,whereas the original lactonase and esterase activities decreased dramatically.A 1.4 × 107-fold of specificity inversion was achieved by only two residue substitutions.Significantly,the thermostability of the variant was not compromised,showing a high T5015 value of 76.5?.New homologous OPHs were further discovered based on the simple double mutation.Among them,PPOPHM2 from Pseudomonas putida emerged as a new promising OPH with a very high specific activity(41.0 U·mg-1)toward methyl-parathion.To elucidate the mechanism for the significantly impoved PTE activity,the crystal structure of PoOPHM2 was determined at 2.25 A resolution.Docking study suggested that mutations H250I and 1263 increase the hydrophobic interaction of the enzyme with substrate,facilitating methyl-parathion positioning and binding.Kinetic parameters and evolutionary analysis indicate that it is likely that the initial mutation occurs at position Ile263 for higher PTE activity,and the later tuning occurs at His250 which results in the loss of lactonase activity.Furthermore,we enhanced the degradation rate of malathion starting with a phosphotriesterase PoOPHM2 while also considering the thermostability using a newly designed Hierchachical Iteration Mutagenesis(HIM)strategy.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),the thermostability of 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 the parental PoOPHM2,with a thermostability(T5015)of 67.6 ?.Therefore,PoOPHM9 appears to be an efficient and robust candidate for malathion detoxification.The expression level of the malathion degrader was improved by engineering the recombinant expression host.After condon optimization and removing native signal peptide,the secretory expression of PoOPHM9 was achieved in P.pastoris.In the optimized pH,promoter and carbon source conditions,the constitutive expression strain X33/pGAPZaA-?poophm9 could reach a protein yield of 4.1 g/1 or a volumetric productivity of 640 U/(1·h)using either glucose or glycerol as carbon source,which is significantly higher than the level of recombinant E.coli fermentation.The selected recombinant yeast was cultivated with convenient fermentation process,simple medium component,no addition of methanol and displayed no degradation of the target protein.It showed some promise for an easy,efficient and economic route for the enzyme production.Lastly,three kinds of immobilized enzymes were prepared as a malathion degrader.As a result,CLEA-?PoOPHM9 displayed the highest specific acticity and activity recovery.APoOPHM9@ESR-2 was among the most stable enzymes,which did not lose any activity after incubating at 30? for 30 days or being reused for 10 times.?PoOPHM9 alingate capsules could completely degrade malathion due to its lower KM value.The three immobilized enzymes show the various particle sizes and diverse advantages which can meet the ditoxification requirements for different occasions.