Shreya thesis

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Shreya thesis

  1. 1. AMIDOHYDROLASES PROFILINGFROM SOILS OF NORTH GUJARAT Guided & Checked By : Dr. S.A. Bhatt sir Shreya M. Modi MSc Sem-IV Exam No.- 181
  2. 2. SAMPLING SITES
  3. 3. AMIDOHYDROLASE ENZYME• Amidohydrolases are type of hydrolase enzyme that acts upon C-N bond in amide.• They are categorized under EC number EC 3.5.1 and 3.5.2.• They are called deaminase, deamidizing enzymes.• Eg.,• L-asparginase,• L-glutaminase,• Amidase, etc…• Amidohydrolases involve in subsequent ammonification from amino acids to ammonia
  4. 4. INTRODUCTION• Amidase is the enzyme that catalyses the hydrolyses of amides, and Produces Ammonia and the corresponding Carboxylic acid.• Amidase acts on C-N bonds in linear amides.• E.C. No.- 3.5.1.4 RCONH2 + H2O Amidase NH3 + RCOOH
  5. 5. PRINCIPLE• This method involves determination of the NH4 -N released by amidase activity When Soil is incubated with buffered (0.1 M Tris Hydroxy methyl Amino methane) THAM. , (pH 8.5).• The Ammonium released is determined by a rapid procedure involving treatment of the incubated soil sample with 2.5 M KCl Containing an amidase inhibitor (Uranyl acetate) and steam distillation of an aliquot of the resulting suspension with MgO for 3.3 min.
  6. 6. CHEMICALS• Toluene• Tris sulphuric acid buffer (0.1 m, pH 8.5)• Amide solution (0.5 M)• Potassium chloride (2.5 M) – uranyl acetate (0.005 M ) solution• Reagents for determination of Ammonia Magnesium oxide Boric acid indicator solution 0.0025 M H2SO4
  7. 7. REQUIREMENT• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)
  8. 8. PROCEDURETake 50ml volumetric flask Add 5g moist soilAdd 0.2ml Toluene and 9ml Tris buffer Mix it well Add 1 ml 0.5M Amide solution
  9. 9. Mix well for few second Stopper the flask Incubate for 2 hrs at 37◦C Add approximate 35ml KCl-UO2(C2H3O2)2:2H2OSwirl the flask and cool at room temperature
  10. 10. Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O Mix the content thoroughly
  11. 11. PROCEDURE FOR CONTROL Take 50ml volumetric flask Add 5g moist soil Add 0.2ml Toluene and 9ml Tris buffer Mix it well Stopper the flask
  12. 12. Incubate for 2 hrs at 37◦CAdd approximate 35ml KCl-UO2(C2H3O2)2:2H2O Add 1 ml 0.5M Amide solution Swirl the flask and cool at room temperature
  13. 13. Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O Mix the content thoroughly
  14. 14. ESTIMATION OF RELEASED AMMONIA Take a Erlenmeyer flask Pipette 5ml boric acid Put it in its special place Pipette 20ml of the resulting soil suspension into a 100ml distillation flask
  15. 15. Add 0.2g MgO Steam distillate content until 30ml ofdistillate are collected in the flask Titrate the distillate with 0.005M H2SO4
  16. 16. INTRODUCTION• The enzyme L-asparginase has important role in nitrogen mineralization of soil.• L-asparginase activity was first detected by Drobni’k(1956). Some evidence suggest that, a portion of released NH4+ comes from hydrolysis of amide ( Asparginase and glutaminase)residues in soil organic matter.• E.C. No- 3.5.1.1.
  17. 17. Cont….• It catalyses the hydrolysis of L- aspargine, which produce L-aspartic acid and ammonia.
  18. 18. PRINCIPLE• Frankenberger and Tabatabai(1991a) developed a simple, precise and sensitive method to assay L- asparginase activity in soils.• This method uses steam distillation to determine the NH4+ produced by L-asparginase activity when soil is incubated at 37˚ C for 2hrs.• The procedure developed gives quantitative recovery of NH4-N added to soils and does not cause chemical hydrolysis of L-aspargine.
  19. 19. REQUIREMENT• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)
  20. 20. CHEMICALS• Toluene• Tham buffer• KCl-Ag2SO4 solution• 0.5M asparagine solution• MgO• 0.005M H2SO4• Indicator solution
  21. 21. Cont……• Boric acid indicator solution• 0.05M NaOH• Ammonium standard soluton• 95% ehanol• Distiller water
  22. 22. CALCULATION C × 50L-asparginase activity = dwt × 5 Where, C = measured NH4-N mL 1 dwt = dry weight of 1g moist soil 5 = Weight of used soil in test 50 = Total volume of soil suspension
  23. 23. PHYSICOCHEMICAL ANALYSISNo Parameter Method Used1 Colour Munsell’s Soil Colour Chart2 pH pH Meter3 Calcium carbonate Rapid Titration method4 Organic carbon Walkley and Black’s method5 Phosphorus Fiske and Subbarow’s Method6 Sulfur Spectrophotometric method7 Total hardness EDTA titration method8 Inorganic nitrogen Dumas method9 Chloride Mohr’s method10 Bicarbonate Titration method
  24. 24. AMIDASEPLACE 1OCM. μg /1gsoil 20CM. μg/1gsoilHIMMATNAGAR 430 328.57TALOD 260 150MODASA 400 233.33VIJAPUR 950 950VISNAGAR 172.73 28.57TARABH 350 300NEDARA 175.71 225CHANASMA 60 52.5ADIYA 155 51.43 AMIDASE 1000 900 800 700 600 500 400 1OCM. μg /1gsoil 300 20CM. μg /1gsoil 200 100 0
  25. 25. L-ASPARGINASEPLACE 1OCM. μg /1g of soil 20CM. μg /1g of soilHIMMATNAGAR 336 5.72TALOD 84 46.67MODASA 566.67 340VIJAPUR 330 55VISNAGAR 95 194.29TARABH 250 100NEDARA 84.5 69CHANASMA 102.86 60ADIYA 220 154.29 L-ASPARGINASE600500400300200 1OCM. μg /1gsoil100 20CM. μg /1gsoil 0
  26. 26. COLOUR DISTRICT PLACE COLORSABARKANTHA HIMATNAGAR DARK BLACK TALOD BROWN MODASA BROWN MEHSANA VIJAPUR BROWN BLACK VISNAGAR BLACK TARABH BROWNISH YELLOW PATAN NEDARA BROWN BLACK CHANASMA BLACK ADIYA BROWNISH YELLOW
  27. 27. pH DISTRICT PLACE 10cm,pH 20cm,pH HIMATNAGAR 8.5 7.5 SABARKANTHA TALOD 7 7.5 MODASA 7 7 VIJAPUR 7.5 7.5 MEHSANA VISNAGAR 8 8 TARABH 7.5 8 NEDARA 8 7.5 PATAN CHANASMA 8 7.5 ADIYA 8 7.5 pH98765 10CM,Ph43 20CM,Ph210 HIMATNAGAR TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA
  28. 28. INORGANIC NITROGEN DISTRICT PLACE 10CM, μg/ml 20CM, μg/mlSABARKANTHA HIMATNAGAR 0.222 0.825 TALOD 0.412 0.349 MODASA 0.38 0.38 MEHSANA VIJAPUR 1.174 0.73 VISNAGAR 0.38 0.666 TARABH 0.317 0.158 PATAN NEDARA 1.17 0.73 CHANASMA 1.71 1.96 ADIYA 2.53 2.15 INORGANIC NITROGEN 32.5 21.5 1 10CM, μg/ml 20CM, μg/ml0.5 0
  29. 29. SULFUR DISTRICT PLACE 10CM, mg/lit 20CM, mg/ltSABARKANTHA HIMATNAGAR 40 60 TALOD 86.66 100 MODASA 73.33 46.66 MEHSANA VIJAPUR 73.33 53.33 VISNAGAR 86.66 60 TARABH 53.33 53.33 PATAN NEDARA 40 53.33 CHANASMA 41.76 38.51 ADIYA 52.27 46.13 SULFUR120100806040 10CM, mg/lit20 20CM, mg/lt 0
  30. 30. CARBON DISTRICT PLACE 10CM,mg/lit, 20CM, mg/lit,SABARKANTHA HIMATNAGAR 125.2 107.8 TALOD 94.6 109.4 MODASA 113.8 114.6 MEHSANA VIJAPUR 104.6 107.4 VISNAGAR 95 106.8 TARABH 125.8 131.8 PATAN NEDARA 112.2 102.8 CHANASMA 133.2 118.6 ADIYA 113.6 131 CARBON1401201008060 10CM,mg/lit40 20CM, mg/lit20 0
  31. 31. CHLORIDE DISTRICT PLACE 10CM, mg/100g 20CM, mg/100 g SABARKANTHA HIMATNAGAR 39.4 55.38 TALOD 31.95 35.5 MODASA 24.49 31.95 MEHSANA VIJAPUR 24.85 46.15 VISNAGAR 31.95 29.11 TARABH 56.8 35.14 PATAN NEDARA 56.09 39.05 CHANASMA 49.7 39.76 ADIYA 42.6 35.5 CHLORIDE6050403020 10CM, mg/100g 20CM, mg/100g10 0
  32. 32. CALCIUM CARBONATE DISTRICT PLACE 10CM ,in% 20CM ,in% SABARKANTHA HIMATNAGAR 3.5 3 TALOD 2.5 2.5 MODASA 3 2 MEHSANA VIJAPUR 1 0.5 VISNAGAR 5 5.5 TARABH 2 1.5 PATAN NEDARA 3 3 CHANASMA 4.5 5.5 ADIYA 3.5 4 CALCIUM CARBONATE65432 10CM ,in%1 20CM ,in%0
  33. 33. TOTAL HARDNESS DISTRICT PLACE 10CM, mg/lit 20CM, mg/lit SABARKANTHA HIMATNAGAR 940 920 TALOD 700 660 MODASA 700 660 MEHSANA VIJAPUR 280 800 VISNAGAR 940 800 TARABH 800 1300 PATAN NEDARA 640 700 CHANASMA 1000 3500 ADIYA 700 1240 TOTAL HARDNESS400035003000250020001500 10CM, mg/lit1000 500 20CM, mg/lit 0
  34. 34. CARBONATE DISTRICT PLACE 10CM,mg/100g 20CM,mg/100gSABARKANTHA HIMATNAGAR 12 10.5 TALOD 9.3 7.5 MODASA 10.5 7.5 MEHSANA VIJAPUR 10.5 6 VISNAGAR 12 9 TARABH 35.1 12 PATAN NEDARA 7.5 9 CHANASMA 6.3 12 ADIYA 6 7.5 CARBONATE4035302520 10CM,mg/100g15 20CM,mg/100g1050 HIMATNAGAR TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA
  35. 35. BICARBONATE DISTRICT PLACE 10CM,mg/100g 20CM,mg/100gSABARKANTHA HIMATNAGAR 12.2 9.15 TALOD 6.1 9.15 MODASA 18.3 9.15 MEHSANA VIJAPUR 24.4 9.15 VISNAGAR 12.2 12.2 TARABH 18.3 15.25 PATAN NEDARA 12.2 21.35 CHANASMA 15.25 10.67 ADIYA 24.4 21.35 BICARBONATE3025201510 10CM,mg/100g 5 20CM,mg/100g 0
  36. 36. CONCLUSION• According to the data of Amidase enzyme activity, all samples of soil give high concentration of Amidase in 10cm depth expect Nedra from Patan district..• In the case of L-Asparginase activity samples given least varied in 10cm depth.• Amidase and L-Asparginase enzyme activity shown decreases as depth increases.• Soil analysis data from Carbon, Chloride, Carbonate, Sulfur, Bi-Carbonate, etc… are in high amount in most of all samples of soil.
  37. 37. REFERENCES• Nannipieri, P., E. Kandeler and P. Ruggiero. (2002). Enzyme activities and microbiological and biochemical processes in soil. p. 7–8. In R.G. Burns and R.P. Dick (ed.) Enzymes in the environment: Activity, ecology, and applications. Marcel Dekker, New York.• Tabatabai MA, Bremner JM (1971). Michaelis constants of soil enzymes. Soil Biol. Biochem. 3: 317- 323.• APHA, Standard Methods for Water and Waste Water Analysis, New York. (1992)
  38. 38. Thank You

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