methyl parathion hydrolase

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Methyl parathion hydrolase producing fungi


  • 1. ISOLATION & SCREENING OF METHYL PARATHIONDEGRADING ASPERGILLI FROM SOILAProject Report Submitted In Part Fulfilment Of The RequirementFor The Degree Of Masters Of ScienceInApplied Microbiology & Biotechnology By Akanksha Khare Department of Applied Microbiology and BiotechnlogyDr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P) 470 003

2. IntroductionIntroductionPesticides are Xenobiotics i.e,man made compound with structure thatmicroorganism have never been exposed to. Many of these arerecalcitrant i.e remaining unchanged in environment. Methyl parathion (O,O-dimethyl, O-p nitrophenol phosphorothioate) isa broad spectrum organophosphorus insecticides has molecularformula of C8H10NO5PS, with molecular mass of 263.23 daltons.Pure methyl parathion is white crystalline, solid powder whichsolubility 55 60 mg/ liter at 25C in water.Soil microbes convert MP into dimethyl thiophosphoric acid and p nitrophenol (pnp) by hydrolysis. 3. Mechanism of ToxicityMechanism of toxicityThe primary effect associated with high level exposure to OP insecticides isinhibition of acetylcholinesterase (AChE), resulting in acetylcholineaccumulation.Resulting in excessive nervous stimulation culminating in respiratoryfailure & death. Hypotension, Bradycardia, Bronchoconstriction &Bronchial fluid accumulation, symptoms that results from the inability ofrespiratory muscles to work. Clinical symptoms of poisoning with MP include pallor, sweating,dizziness, vomiting, diahorrhea, abdominal cramps, headache, blurredvision, convulsion, dialation of pupils, tears, & cardia arrest. It is easily absorbed via all routes of exposure (oral, dermal or inhalation)and is easily distributed to the tissues of body.p nitrophenol, the other hydrolysis product of MP is toxic to plant,animals and human. It is also a suspected carcinogen 4. Biodegradation of MPBiodegradation of MPHydrolysis is the principle mechanism of biodegradation of pesticides.Complete degradation of which occurs when its hydrolysis productsenters into krebs cycle.Many bacterial species like Pseudomonas, Alkaligenes, Acinetobacter,and Arthrobacter has been reported to detoxify most of pesticides, butlittle is known about role of fungi for the same.White rot fungi like Trametes versicolor, Phanerochaete chrysosporiumetc are known for partial degradation of pesticides.The whole process is carried out by Methyl parathion hydrolase (MPH).MPH belongs to metallolactamase superfamily and has high catalyticactivity towards Organophosphates among all organophosphorushydrolase (OPH) 5. Mode action of MPH and further metabolic activitiesMode of of Action of MPH & furthermetabolism of MP 6. -ApplicationsBioremediation In biodegradation of organophosphorus pesticides.A company in Australia now sells carrier based OPH enzymes forremoval of OP from Sheep-dip water before it can be applied soil.Biotehnological It is succesfully used to develop and evaluatebiosensors and Organophosphorus contamination. 7. Key objectives of the study To isolate & screen Methyl parathion Hydrolase producing Aspergilli. To compare the potential of seven different Aspergillus nigertowards Methyl Parathion degradation and Methyl parathion Hydrolaseproduction. To quantify the production of methyl parathion hydrolase, bydetermining enzyme activity. 8. Materials & MethodsIsolation of FungiFor isolation of fungi, Saboraud Dextrose Agar media was used.CompositionDextrose-40.0 gmPeptone -10.0 gmAgar agar -20.0gmDistilled water- 1000ml pH- 7.0 Method used - Direct plate method 9. 1. Garden soil(Dept. of2 GSM1 A.niger microbiology) GSM2 A.fumigatus2.Garden soil(Botany 3 GSB1 A.nigerDept.) GSB2 A.fumigatus GSB3A.terreus3. Chemistry Dept.(Near3 CLS1A.flavipeslab. discharge)CLS2 A.niger CLS3 A.fumigatus4. Compost soil2 CS2A.versicolor CS3A.niger5.Agricultural 3 ASM1 A.niger soil(Makroniya),sagar ASM2 A.versicolor ASM3 A.fumigatus6.Agricultural 3 ASK1A.flavussoil(Kanera),Sagar ASK2A.terreus ASK3 A.fumigatus7.Agricultural 4 ASP4 A.nigersoil(Pathriya),sagar ASP1A.tamari ASP2 A.glaucus ASP3 A.melleus 10. Primary screeningAspergillus versicolor Aspergillus glaucus 11. Aspergillus niger Aspergillus melleusAspergillus ustusAspergillus terreus 12. Screeening of MPH in BrothScreenig for MPH Production in Broth. Media- Czapeks Dox Broth(With out sucrose) NaNO3- 2.0 gm KCl- 0.5 gm MgSO4.H2O- 0.5 gm FeSO4.7H2O- TraceK2HPO4-1 gmTween 80 - 4 mlDistilled water -1000 mlVishniac Solution (gm/ltr) Contain EDTA (10), ZnSo4.7H2O (4.40),CaCl2.2H2O (1.47). It is used particularly to enhance the growth ofAspergilli.Concentration of MP(As carbon Source)Concentration of Methyl parathion were prepared in ppm.Fourconcentrations i.e., 15 ppm(0.15 mg in 100 ml Distilled water) ,10 ppm(0.1 mg in 100 ml distilled water),20ppm(0.20mg in 100ml distilledwater) & 30 ppm. 13. 10 ppmAspergillus versicolorAspergillus nigerAspergillus terreus 14. 15 ppmAspergillus terreus Aspergillus versicolorAspergillus niger 15. -Methodology50 ml of media were taken in well labelled 150 ml Erlenmeyer flaskeach for 10 ppm & 15 ppm concentration of Methyl parathion.Three disc of each fungal isolate were inoculated in respective flasks.One one control for each concentration was also prepared having onlymedia but no fungal discs.Flasks now kept in shaker at 28C, 120 rpm for 7 days.Assay of Methyl Parathion Hydrolase(MPH Activity)Release of para - nitrophenol,an indication of hydrolysis of methylparathion was taken as a criterion for the estimation of production ofmethyl parathion hydrolase ,which was assayed spectrophotometricallyat 405 nm(Absorbance of PNP). 16. Preparations For MPH ActivityCitrate buffer (0.05 M,pH 5)-1.05 gm of citric acid was dissolved in 100ml of distilled water. Adjust pH 5 with 0.2 M NaOH.Substrate solution-50 mg of methyl parathion were dissolved in 50 mldistilled water.Stopping reagent (1.0 M Na2CO3)-Dissolve 10.6 gm of Na2CO3 in 100 mlof deionized water.Standard solution (Stock)-0.0695 pnp (0.01 M) taken in 50 ml ofvolumetric flask & filled up to mark with citrate buffer.Preparations of dilutions : DilutionsConcentration(mol/ml)Concentration(nkats/ml)1:200.500.8331:50 0.200.3331:100 0.10 0.1671:200 0.05 0.083 17. BlankStandardsEnzyme BlankReaction TubeAdd 1.8 ml ofAdd 1.8 ml ofAdd 1.8 ml ofAdd 1.8 ml ofsubstrate solution substrate solution substate solutionsubstrate solutionIncubate for 60 50C0.2 ml Cultural Filterate0.2 ml Buffer0.2 ml dilutionsIncubate for 60Incubate for 60min. at 50C0.2 ml stoppingIncubate for 60 min. atmin. at 50CReagent50C0.2 ml stopping0.2 ml stopping0.2 ml CulturalReagentReagent 0.2 ml stoppingFilterateReagentVortex & take Absorbance at 405 nm 18. Dilutions Concentration Absorbance (mol/ml)(405nm) 1:2000.050.057 1:100 0.10.1351:50 0.20.2461:20 0.50.631Table- Readings for Standard Curve of PNP 19. Standard Curve Of para-nitrophenol0.70.6y = 1.262x - 0.001Absorbance(405nm)0.5R = 0.9990.4Absorbance(405nm)0.30.2Linear (Absorbance(405nm)0.1) 00 0.2 0.40.6Concentration(mol/ml) 20. ORGANISM ENZYME ACTIVITY10PPM 15 PPM A.flavus NILNILA.flavipesNILNIL A.fumigatus O.145 NILA.glaucus0.7360.503A.melleus1.2130.937 A.nidulans NILNIL A.niger 4.0213.869A.penicilloides NILNILA.tamariNILNILA.terreus3.5463.286 21. MPH ACTIVITY OF ASPERGILLI4.5ENZYME ACTIVITY 43.5 32.5 21.5 10.5 0 FUNGAL ISOLATES 22. Percentage of MP degradationAfter determining Enzyme activity in culture filtrate of TestAspergilli, percentage of Methyl Parathion was also calculated. The calculation was done by using following formula:Percentage of Degradation= [1- Absorbance of test sample/Absorbance ofcontrol] 100 23. 10PPM 15PPM A.flavusNIL NILA.flavipes NIL NIL A.fumigatus NIL NILA.glaucus43.538.3A.melleus66.239.4 A.nidulansNIL NIL A.niger 85.667.1A.penicilloidesNIL NILA.tamari NIL NILA.terreus75.363.6 24. PERCENTAGE OF MP DEGRADATION 90 80PERCENTAGEOF DEGRADATION 70 60 50 40 10PPM 30 15PPM 20 10 0 FUNGAL ISOLATES 25. Screening of Different A.niger for MPHactivityAfter determining the MPH activity & percentage of MP degradation indifferent Aspergilli, it was noted that only A.niger gave maximumenzyme activity & percentage of MP degradation in bothconcentrations.Thus it was supposed to be valuable to screen different A.nigerisolated from different sources. 26. Source of IsolationIsolate numberEnzyme Activity(nkats/ml)30 ppm 20ppmGround nut seedA.N-12.0833.670Compost soil A.N-22.1174.221Botanical Garden soilA.N-31.7333.369Agricultural soil (Kanera) A.N-44.9126.542Agricultural soil(Makroniya)A.N-52.4673.503Chemistry Deptt. SoilA.N-62.4133.119Agricultural soil (Pathriya) A.N-73.72O3.787 27. MPH Activity of different A.nigers765Enzyme activity43 30 ppm20 ppm210A.N-1 A.N-2 A.N-3 A.N-4 A.N-5 A.N-6 A.N-7 Isolate numbers 28. DiscussionOn screening the different Aspergilli, maximum MPH activity showed byA.niger in both 15ppm &10ppm concentration.While A.terreus, A.versicolor, A.melleus, A.ustus & A. glaucus, showedless MPH activity in 15 ppm & more activity in 10 ppm. A.fumigatus gaveless activity in 10 ppm but no activity in 15 ppm.Where as no activity was found in A.flavus, A.flavipes,A.penicilloides, A.nidulans & A.tamarii.From all the above results, Aspergillus niger was found as potent MPHproducer even at Higher concentration(20 & 30 ppm) of MP. 29. THANK yoU