![Percentage of MP degradation
•After determining Enzyme activity in culture filtrate of Test
Aspergilli, percentage of Methyl Parathion was also calculated.
• The calculation was done by using following formula:
Percentage of Degradation= [1- Absorbance of test sample/Absorbance of
control] × 100](https://image.slidesharecdn.com/akakshapowerpoint-120723122905-phpapp02/85/Methyl-parathion-hydrolase-22-320.jpg)
Methyl parathion hydrolase
- 1. ISOLATION & SCREENING OF METHYL PARATHION DEGRADING ASPERGILLI FROM SOIL A Project Report Submitted In Part Fulfilment Of The Requirement For The Degree Of Masters Of Science In Applied Microbiology & Biotechnology By Akanksha Khare Department of Applied Microbiology and Biotechnlogy Dr. Hari Singh Gour Vishwavidyalaya, Sagar (M.P) 470 003
- 2. Introduction Introduction •Pesticides are Xenobiotics i.e,man made compound with structure that microorganism have never been exposed to. Many of these are recalcitrant i.e remaining unchanged in environment. • Methyl parathion (O,O-dimethyl, O-p nitrophenol phosphorothioate) is a broad spectrum organophosphorus insecticides has molecular formula of C8H10NO5PS, with molecular mass of 263.23 daltons. •Pure methyl parathion is white crystalline, solid powder which solubility 55 – 60 mg/ liter at 25˚C in water. •Soil microbes convert MP into dimethyl thiophosphoric acid and p – nitrophenol (pnp) by hydrolysis.
- 3. Mechanism of Toxicity Mechanism of toxicity •The primary effect associated with high level exposure to OP insecticides is inhibition of acetylcholinesterase (AChE), resulting in acetylcholine accumulation. •Resulting in excessive nervous stimulation culminating in respiratory failure & death. Hypotension, Bradycardia, Bronchoconstriction & Bronchial fluid accumulation, symptoms that results from the inability of respiratory muscles to work. • Clinical symptoms of poisoning with MP include pallor, sweating, dizziness, vomiting, diahorrhea, abdominal cramps, headache, blurred vision, 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 MP Biodegradation of MP •Hydrolysis is the principle mechanism of biodegradation of pesticides. Complete degradation of which occurs when its hydrolysis products enters into krebs cycle. •Many bacterial species like Pseudomonas, Alkaligenes, Acinetobacter, and Arthrobacter has been reported to detoxify most of pesticides, but little is known about role of fungi for the same. •White rot fungi like Trametes versicolor, Phanerochaete chrysosporium etc 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 catalytic activity towards Organophosphates among all organophosphorus hydrolase (OPH)
- 5. Mode action of MPH and further metabolic activities Mode of of Action of MPH & further metabolism of MP
- 6. - Applications •Bioremediation – In biodegradation of organophosphorus pesticides. A company in Australia now sells carrier based OPH enzymes for removal of OP from Sheep-dip water before it can be applied soil. •Biotehnological – It is succesfully used to develop and evaluate biosensors 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 niger towards Methyl Parathion degradation and Methyl parathion Hydrolase production. To quantify the production of methyl parathion hydrolase, by determining enzyme activity.
- 8. Materials & Methods Isolation of Fungi For isolation of fungi, Saboraud Dextrose Agar media was used. Composition Dextrose - 40.0 gm Peptone - 10.0 gm Agar – agar - 20.0gm Distilled water - 1000ml pH - 7.0 • Method used - Direct plate method
- 9. 1. Garden soil(Dept. of 2 GSM1 A.niger microbiology) GSM2 A.fumigatus 2. Garden soil(Botany 3 GSB1 A.niger Dept.) GSB2 A.fumigatus GSB3 A.terreus 3. Chemistry Dept.(Near 3 CLS1 A.flavipes lab. discharge) CLS2 A.niger CLS3 A.fumigatus 4. Compost soil 2 CS2 A.versicolor CS3 A.niger 5. Agricultural 3 ASM1 A.niger soil(Makroniya),sagar ASM2 A.versicolor ASM3 A.fumigatus 6. Agricultural 3 ASK1 A.flavus soil(Kanera),Sagar ASK2 A.terreus ASK3 A.fumigatus 7. Agricultural 4 ASP4 A.niger soil(Pathriya),sagar ASP1 A.tamari ASP2 A.glaucus ASP3 A.melleus
- 10. Primary screening Aspergillus versicolor Aspergillus glaucus
- 11. Aspergillus niger Aspergillus melleus Aspergillus ustus Aspergillus terreus
- 12. Screeening of MPH in Broth Screenig for MPH Production in Broth . • Media- Czapek’s Dox Broth(With out sucrose) NaNO3 - 2.0 gm KCl - 0.5 gm MgSO4.H2O - 0.5 gm FeSO4.7H2O - Trace K2HPO4 - 1 gm Tween 80 - 4 ml Distilled water - 1000 ml •Vishniac Solution (gm/ltr) – Contain EDTA (10), ZnSo4.7H2O (4.40), CaCl2.2H2O (1.47). It is used particularly to enhance the growth of Aspergilli. •Concentration of MP(As carbon Source) Concentration of Methyl parathion were prepared in ppm.Four concentrations 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 distilled water) & 30 ppm.
- 13. 10 ppm Aspergillus versicolor Aspergillus niger Aspergillus terreus
- 14. 15 ppm Aspergillus terreus Aspergillus versicolor Aspergillus niger
- 15. - Methodology •50 ml of media were taken in well labelled 150 ml Erlenmeyer flask each 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 only media but no fungal discs. •Flasks now kept in shaker at 28˚C, 120 rpm for 7 days. Assay of Methyl Parathion Hydrolase(MPH Activity) •Release of para - nitrophenol,an indication of hydrolysis of methyl parathion was taken as a criterion for the estimation of production of methyl parathion hydrolase ,which was assayed spectrophotometrically at 405 nm(Absorbance of PNP).
- 16. Preparations For MPH Activity •Citrate buffer (0.05 M,pH 5)-1.05 gm of citric acid was dissolved in 100 ml of distilled water. Adjust pH 5 with 0.2 M NaOH. •Substrate solution-50 mg of methyl parathion were dissolved in 50 ml distilled water. •Stopping reagent (1.0 M Na2CO3)-Dissolve 10.6 gm of Na2CO3 in 100 ml of deionized water. •Standard solution (Stock)-0.0695 pnp (0.01 M) taken in 50 ml of volumetric flask & filled up to mark with citrate buffer. •Preparations of dilutions :– Dilutions Concentration(µmol/ml) Concentration(nkats/ml) •1:20 0.50 0.833 •1:50 0.20 0.333 •1:100 0.10 0.167 •1:200 0.05 0.083
- 17. Blank Standards Enzyme Blank Reaction Tube Add 1.8 ml of Add 1.8 ml of Add 1.8 ml of Add 1.8 ml of substrate solution substrate solution substate solution substrate solution Incubate for 60 min. at 50˚C 0.2 ml Cultural Filterate 0.2 ml Buffer 0.2 ml dilutions Incubate for 60 Incubate for 60 min. at 50˚C 0.2 ml stopping Incubate for 60 min. at min. at 50˚C Reagent 50˚C 0.2 ml stopping 0.2 ml stopping 0.2 ml Cultural Reagent Reagent 0.2 ml stopping Filterate Reagent Vortex & take Absorbance at 405 nm
- 18. Dilutions Concentration Absorbance (µmol/ml) (405nm) 1:200 0.05 0.057 1:100 0.1 0.135 1:50 0.2 0.246 1:20 0.5 0.631 Table- Readings for Standard Curve of PNP
- 19. Standard Curve Of para-nitrophenol 0.7 0.6 y = 1.262x - 0.001 Absorbance(405nm) 0.5 R² = 0.999 0.4 Absorbance(405nm) 0.3 0.2 Linear (Absorbance(405nm) 0.1 ) 0 0 0.2 0.4 0.6 Concentration(µmol/ml)
- 20. ORGANISM ENZYME ACTIVITY 10PPM 15 PPM A.flavus NIL NIL A.flavipes NIL NIL A.fumigatus O.145 NIL A.glaucus 0.736 0.503 A.melleus 1.213 0.937 A.nidulans NIL NIL A.niger 4.021 3.869 A.penicilloides NIL NIL A.tamari NIL NIL A.terreus 3.546 3.286
- 21. MPH ACTIVITY OF ASPERGILLI 4.5 ENZYME ACTIVITY 4 3.5 3 2.5 2 1.5 1 0.5 0 FUNGAL ISOLATES
- 22. Percentage of MP degradation •After determining Enzyme activity in culture filtrate of Test Aspergilli, percentage of Methyl Parathion was also calculated. • The calculation was done by using following formula: Percentage of Degradation= [1- Absorbance of test sample/Absorbance of control] × 100
- 23. 10PPM 15PPM A.flavus NIL NIL A.flavipes NIL NIL A.fumigatus NIL NIL A.glaucus 43.5 38.3 A.melleus 66.2 39.4 A.nidulans NIL NIL A.niger 85.6 67.1 A.penicilloides NIL NIL A.tamari NIL NIL A.terreus 75.3 63.6
- 24. PERCENTAGE OF MP DEGRADATION 90 80 PERCENTAGEOF DEGRADATION 70 60 50 40 10PPM 30 15PPM 20 10 0 FUNGAL ISOLATES
- 25. Screening of Different A.niger for MPH activity •After determining the MPH activity & percentage of MP degradation in different Aspergilli, it was noted that only A.niger gave maximum enzyme activity & percentage of MP degradation in both concentrations. •Thus it was supposed to be valuable to screen different A.niger isolated from different sources.
- 26. Source of Isolation Isolate number Enzyme Activity(nkats/ml) 30 ppm 20ppm Ground nut seed A.N-1 2.083 3.670 Compost soil A.N-2 2.117 4.221 Botanical Garden soil A.N-3 1.733 3.369 Agricultural soil (Kanera) A.N-4 4.912 6.542 Agricultural soil (Makroniya) A.N-5 2.467 3.503 Chemistry Deptt. Soil A.N-6 2.413 3.119 Agricultural soil (Pathriya) A.N-7 3.72O 3.787
- 27. MPH Activity of different A.nigers 7 6 5 Enzyme activity 4 3 30 ppm 20 ppm 2 1 0 A.N-1 A.N-2 A.N-3 A.N-4 A.N-5 A.N-6 A.N-7 Isolate numbers
- 28. Discussion •On screening the different Aspergilli, maximum MPH activity showed by A.niger in both 15ppm &10ppm concentration. •While A.terreus, A.versicolor, A.melleus, A.ustus & A. glaucus, showed less MPH activity in 15 ppm & more activity in 10 ppm. A.fumigatus gave less 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 MPH producer even at Higher concentration(20 & 30 ppm) of MP.
- 29. THANK yoU