Activity Improvement of Organophosphorus Hydrolase Enzyme by Error Prone PCR Method

Pesticides are categorized into the most toxic substances that greatly diffuse in our environment and have high risks for humans and animals. Organophosphates (OPs) are one of the most harmful pesticides that are used in agriculture, household, gardens, domestic animals, sheep dips and even as chemical warfare.1,2 Malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, soman, sarin, tabun, VX, tribufos are different types of OPs.3 These compounds can cause respiratory failure and death (at sufficient dosage) in vertebrates by phosphorylation of the acetylcholinesterase enzyme and loss of its function.1 Thus, the development of materials for bioremediation of OP compounds is essential. Some of the bacteria e.g. Flavobacterium sp.,4 Pseudomonas sp.,5,6 Agrobacterium radiobacter, Enterobacter sp.7-9 and also some of the bacterial enzymes include organophosphorus hydrolase (OPH),7 methyl parathion hydrolase (MPH),10 organophosphorus acid anhydrolase (OPAA)11,12 are able to degrade one or more OP compounds. So far, bioremediation of OPs has been studied. For example, in our pervious study, the mutants of OPH enzyme were designed, carrying disulfide bridges in their structure. These mutants improved the thermostability of OPH enzyme but they showed no improvement in biodegradation activity.13 In order to create an environment for decontamination of OP compounds, the strains and their enzymes should own high and satisfactory biodegradation activity. The directed evolution can offer superior properties to enzymes to improve their activity. Among different methods of directed evolution, the Site-directed mutagenesis of OPH7,14,15 and DNA shuffling15,16 were successfully applied to increase the enzyme efficiency. In this study, we tried to gain recombinant OPH that is able to degrade diazinon (as its only carbon, phosphorus and energy source) more efficiently than native OPH by using error prone PCR method.


Introduction
Pesticides are categorized into the most toxic substances that greatly diffuse in our environment and have high risks for humans and animals.Organophosphates (OPs) are one of the most harmful pesticides that are used in agriculture, household, gardens, domestic animals, sheep dips and even as chemical warfare. 1,2Malathion, parathion, diazinon, fenthion, dichlorvos, chlorpyrifos, soman, sarin, tabun, VX, tribufos are different types of OPs. 3 These compounds can cause respiratory failure and death (at sufficient dosage) in vertebrates by phosphorylation of the acetylcholinesterase enzyme and loss of its function. 1hus, the development of materials for bioremediation of OP compounds is essential.Some of the bacteria e.g.8][9] and also some of the bacterial enzymes include organophosphorus hydrolase (OPH), 7 methyl parathion hydrolase (MPH), 10 organophosphorus acid anhydrolase (OPAA) 11,12 are able to degrade one or more OP compounds.
So far, bioremediation of OPs has been studied.For example, in our pervious study, the mutants of OPH enzyme were designed, carrying disulfide bridges in their structure.These mutants improved the thermostability of OPH enzyme but they showed no improvement in biodegradation activity. 13n order to create an environment for decontamination of OP compounds, the strains and their enzymes should own high and satisfactory biodegradation activity.
The directed evolution can offer superior properties to enzymes to improve their activity.Among different methods of directed evolution, the Site-directed mutagenesis of OPH 7,14,15 and DNA shuffling 15,16 were successfully applied to increase the enzyme efficiency.
In this study, we tried to gain recombinant OPH that is able to degrade diazinon (as its only carbon, phosphorus and energy source) more efficiently than native OPH by using error prone PCR method.

Enzymes and Reagents
Restriction endonucleases, dACTP, dTTP, dCTP, and dGTP were purchased from bioscience (Germany) and Genetbio (South Korea), respectively.MgCl2 and MnCl2 were obtained from Merck (Germany).T4 DNA ligase and Taq DNA polymerase were purchased from Fermentas to be used in PCR reaction Diazinon as the substrate was prepared by Sigma-Aldrich Co.
Mutagenesis by Error-Prone Polymerase Chain Reaction Random mutations were introduced to wild type oph gene by error-prone polymerase chain reaction (epPCR) method.The conditions for error-prone PCR were designed to create mutant libraries which include less and adequate change in DNA structure.Each 25 µL error prone PCR mixture contained 40 ng of oph gene as a template, 0.4 µM each of the primer OPH-F (5´-ATAAGCTTTCTATCGGCACTGGTGA-3´) and OPH-R (5´-TTCTCGAGGCTTGCACGCAGAGTC-3´), 5.5 mM MgCl2, 0.25 mM MnCl2, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, 1 mM dTTP, 10× PCR buffer without MgCl2 and 5 units of Taq DNA polymerase.The error prone PCR was performed in 30 cycles, with initial denaturation temperature at 94°C for 5 min, followed by 30 cycles of 94°C for 30 seconds, annealing step at 56C for 40 seconds, extending stage at 72°C for 1 minute, and final extension 72°C for 10 minutes.PCR was performed by thermocycler (Eppendorf, North America).The error prone PCR product was observed using agarose gel electrophoresis to determine molecular mass followed by purifying step using QIAquick PCR Purification Kit (Qiagen).

Construction of Expression Library
The purified epPCR product and pET-28a were digested by ZohI and HindIII.After digestion, the cleaved products were ligated by T4 ligase at 16°C overnight to construct plasmid owning mutant gene.The plasmids were cloned into T7 shuffled to assemble expression library.The cloned strains were cultured on LB agar containing kanamycin (40 mg/mL) at 37°C overnight. 19reening All of recombinant strains on LB agar were transferred into LB broth and incubated at 37°C overnight.Then, the recombinant strains (100 µL) were inoculated into 5 mL fresh media until the OD at 600 nm was reached between 0.5-0.7.Clones were induced by 50 mM IPTG and incubated for 4 hours at 37°C. 20After centrifugation, precipitated bacterial cell were transferred on MSM agar (0.1 g/L NaCl, 0.2 g/L KCl, 0.5 g/L (NH4)2SO4, 50 mg/L CaCl2•H2O, 15 g/L agar) supplemented with 5, 50 and 100 mg of diazinon as only carbon, phosphorus and energy source and incubated for 4 days at 37°C.The colonies on MSM agar were subcultured on LB agar and then 20 colonies were selected for assessment of whole cell activity.

Whole Cell Activity
The screened strains were cultured on MSM broth (0.1 g/L NaCl, 0.2 g/L KCl, 0.5 g/L (NH4)2SO4, 50 mg/L CaCl2•H2O) supplemented with 50 mg of diazinon as only carbon, phosphorus and energy source and then incubated for 48 hours at 37°C.Degradation of diazinon (which is measured by concentration of diazinon) at OD=246 nm was assayed at 0, 24 and 48 hours by spectrophotometry (WSP, England).

Optimization of Mg2+ and Mn2+ Concentration in the
Error-Prone PCR System As previously described by Mao et al, 21 the high Mg 2+ concentration causes the high PCR production with an intense strip but the low Mg 2+ concentration, creates a poor PCR strip, even in the absence of Mn 2+ .Thus, we used 4 combinations of the Mg 2+ and Mn 2+ concentrations.The Mg 2+ and Mn 2+ combinations were included 3 and 0 mmol/L, 5 and 0.1 mmol/L, 6 and 0.25 mmol/L, 7 and 0.4 mmol/L of Mg 2+ and Mn 2+ , respectively.We observed that when the Mg 2+ and Mn 2+ combinations were 3 and 0 mmol/L, 5 and 0.1 mmol/L, 6 and 0.25 mmol/L respectively, the PCR product was relatively high but when the Mg 2+ and Mn 2+ combinations were 7 and 0.4 mmol/L respectively, the PCR product was low (Figure 1).Finally, we selected the 5.5 and 0.2 mmol/L of the Mg 2+ and Mn 2+ combination, respectively.

Screening of the Mutant Library
The derived mutant library of epPCR process was cultured on MSM agar supplemented with 5, 50 and 100 mg of diazinon.The mutant colonies were obtained in all of concentrations of diazinon but the E. coli T7 shuffled without oph gene was not able to grow in any of concentration of diazinon.Due to the small size of the mutant colonies, work continued with, 20 selected colonies from LB agar.

Whole Cell Activity
As shown in Figure 2 and Figure 3, at 24 hours, 15 of screened strains (71% of the total) showed elevated degradation ability than the native strains (T7 shuffled strains containing wildtype OPH in pET32).Six screened strains (29%) showed less ability than the native strains in degradation of diazinon.The E6, E9, E15, E16 and E20 screened strains were characterized as the strains which possess highest improvement in degradation ability, with more than 25% increased efficiency (Figure 4).The E6 strain was found to have highest improvement of degradation, with 29.3% ratio.At 48 hours, the E6 strain had the superiority over the native strain in removing diazinon completely.(Figure 5).

Discussion
Oph gene has been manipulated by numerous different rational and directional methods for stability (pH and thermal) or activity improvement, broading the substrate range, decreasing degradation time and offering the ability of degrading in high concentrations of OPs.In this study, for the first time, we applied error prone PCR method to create mutated E. coli strains with improved oph enzyme activity.][24][25][26] We succeeded to optimize the amount of Mg 2+ and Mn 2+ concentrations for epPCR amplification of oph gene.We selected 5.5 and 0.2 mM from Mg 2+ and Mn 2+ respectively, for high throughput PCR product.In this situation, the amount of

Samples
Biodegredation of diazinon PCR product is high and (based on previous study) mutation rate is probably suitable for creating a large and functional library. 17,21,27When Mg 2+ and Mn 2+ concentrations is higher than 6 and 2.5 mM respectively, epPCR product is very low and on the other hand when Mg 2+ and Mn 2+ concentrations is lower than 5 and 0.1 mM respectively, epPCR product is high but probably mutation rate is very low.Indeed, error rate of Taq DNA polymerase has a significant correlation with content of Mg 2+ and Mn 2+ in epPCR reaction. 28In previous study by Mao and colleagues, the concentrations of Mg 2+ and Mn 2+ for high PCR product and suitable mutated frequency was obtained 3 and 0.4 mM for Mg 2+ and Mn 2+ , respectively 21 which is comparable with our study.It should be noted that high Mg 2+ and Mn 2+ concentrations will cause high error rate and finally produce more unique sequences. 29n our previous study by rational design of oph gene, multiple mutants was designed and evaluated.Some of mutants were improved in terms of thermostability (by introducing disulfide bridge) but we observed no improvement in activity. 13It seems, fewer changes are observed in the rational studies and also less number of functional mutants was achieved.
In our study, 73% of screened strains were capable to degrade diazinon more efficiently compared to native strain.The E6 strain was determined as the most potential strain in biodegradation with 29.3% raising ratio which is comparable with a study by Mao on beta-1,3-1,4-glucanase from Bacillus  altitudinis 21 that resulted in improved activity by 48.6%, and also the study by Liao for Recombinant phyA Mutant Phytase from Aspergillus niger N25 that showed 29% improvement. 30owever, in a study by Loo on Epoxide Hydrolase from A. radiobacter using error-prone PCR and DNA shuffling, the mutants gained up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. 17In the study by Zuo on serine hydroxyl methyl transferase gene from Escherichia coli strain AB90054 using DNA shuffling method, a mutant namely 3E7 showed 8-fold increase in enzyme activity and 41-fold increase in enzyme productivity compared with its wild-type parent. 31This is due to introducing so many changes and recombination in target gene but in many cases, success is not achieved using recombination methods such as DNA shuffling, SHIPREC, SCRATCHY or other similar methods In this study, we observed that the ep6 mutant was able to completely degrade diazinon after 48h.It can be concluded that obtained mutations have made changes in enzyme expressing gene that is responsible for both activity and stability improvement.However, in order to prove that further investigation is needed.
As the enzyme was expressed in the cytoplasm of the E. coli host and it is not plasma-membrane bound or secreted form in this host, the screening is associated with many problems.Due to encountered difficulties, in further studies it is suggested that the oph gene should be expressed in secretory host for example B. subtilis or in the form of plasma-membrane bound in yeast or E. coli.

Conclusions
We obtained the mutants of oph gene that are able to express OPH enzymes with profound activity; therefore we recommend the error PCR methods as a rapid method for improvement of the protein properties .The epPCR method has low complexity in comparison with other common methods and can provide a diverse library including efficient mutants.

Figure 1 .
Figure 1.The epPCR Product of oph Gene on 1% Agarose Gel.The Mg 2+ and Mn 2+ combinations are shown.

Figure 2 .
Figure 2. Comparison of Diazinon Biodegradation in Control and Mutant Strains.The level of diazinon biodegradation in 5 mL of MSM agar supplemented with 50 mg/L diazinon for all samples in two time points (24 and 48 hours) was observed.Absorbance was measured at 248 nm.MSM + diazinon was evaluated for definition of spontaneously degradation; T7: E. coli T7 shuffled strain with no vector and oph gene; Native: E. coli T7 shuffled with parent oph gene along with vector pET32; E1 to E21: E. coli T7 shuffled with mutant oph gene including vector pET28.

Figure 3 .
Figure 3. Biodegradation of Diazinon.The 5 preferable mutants, capable of degrading diazinon more efficiently than other strains.

Figure 5 .
Figure 5. Diazinon Biodegradation at 0, 24 and 48 Hours Intervals.The E6 mutant was able to remove diazinon completely after 48 h.