Download to read offline
Uploaded byShreya Modi
Shreya thesis
The document summarizes the results of profiling amidohydrolase enzymes and analyzing physicochemical properties of soil samples collected from various locations in North Gujarat, India. Amidohydrolase enzymes like amidase and L-asparaginase were assayed in the soil samples. Various physicochemical parameters of the soil like color, pH, organic carbon, nitrogen, sulfur, chloride, calcium carbonate and total hardness were also analyzed. The results showed variation in enzyme activities and physicochemical properties across different sampling locations.
Shreya thesis
- 1. AMIDOHYDROLASES PROFILING FROM SOILSOF NORTH GUJARAT Guided & Checked By : Dr. S.A. Bhatt sir Shreya M. Modi MSc Sem-IV Exam No.- 181
- 2.
- 3. AMIDOHYDROLASE ENZYME • Amidohydrolasesare 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. INTRODUCTION • Amidase isthe 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. PRINCIPLE • This methodinvolves 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. CHEMICALS • Toluene • Trissulphuric 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. 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. PROCEDURE Take 50ml volumetricflask Add 5g moist soil Add 0.2ml Toluene and 9ml Tris buffer Mix it well Add 1 ml 0.5M Amide solution
- 9. Mix well forfew second Stopper the flask Incubate for 2 hrs at 37◦C Add approximate 35ml KCl-UO2(C2H3O2)2:2H2O Swirl the flask and cool at room temperature
- 10. Make final volume50ml by addition of KCl-UO2(C2H3O2)2:2H2O Mix the content thoroughly
- 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. Incubate for 2hrs at 37◦C Add approximate 35ml KCl-UO2(C2H3O2)2:2H2O Add 1 ml 0.5M Amide solution Swirl the flask and cool at room temperature
- 13. Make final volume50ml by addition of KCl-UO2(C2H3O2)2:2H2O Mix the content thoroughly
- 14. ESTIMATION OF RELEASEDAMMONIA 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. Add 0.2g MgO Steam distillate content until 30ml of distillate are collected in the flask Titrate the distillate with 0.005M H2SO4
- 16. INTRODUCTION • The enzymeL-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. Cont…. • It catalysesthe hydrolysis of L-aspargine, which produce L-aspartic acid and ammonia.
- 18. PRINCIPLE • Frankenberger andTabatabai(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. 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. CHEMICALS • Toluene • Tham buffer • KCl-Ag2SO4 solution • 0.5M asparagine solution • MgO • 0.005M H2SO4 • Indicator solution
- 21. Cont…… • Boric acid indicator solution • 0.05M NaOH • Ammonium standard soluton • 95% ehanol • Distiller water
- 22. CALCULATION C × 50 L-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. PHYSICOCHEMICAL ANALYSIS No Parameter Method Used 1 Colour Munsell’s Soil Colour Chart 2 pH pH Meter 3 Calcium carbonate Rapid Titration method 4 Organic carbon Walkley and Black’s method 5 Phosphorus Fiske and Subbarow’s Method 6 Sulfur Spectrophotometric method 7 Total hardness EDTA titration method 8 Inorganic nitrogen Dumas method 9 Chloride Mohr’s method 10 Bicarbonate Titration method
- 24. AMIDASE PLACE 1OCM. μg /1gsoil 20CM. μg/1gsoil HIMMATNAGAR 430 328.57 TALOD 260 150 MODASA 400 233.33 VIJAPUR 950 950 VISNAGAR 172.73 28.57 TARABH 350 300 NEDARA 175.71 225 CHANASMA 60 52.5 ADIYA 155 51.43 AMIDASE 1000 900 800 700 600 500 400 1OCM. μg /1gsoil 300 20CM. μg /1gsoil 200 100 0
- 25. L-ASPARGINASE PLACE 1OCM. μg /1g of soil 20CM. μg /1g of soil HIMMATNAGAR 336 5.72 TALOD 84 46.67 MODASA 566.67 340 VIJAPUR 330 55 VISNAGAR 95 194.29 TARABH 250 100 NEDARA 84.5 69 CHANASMA 102.86 60 ADIYA 220 154.29 L-ASPARGINASE 600 500 400 300 200 1OCM. μg /1gsoil 100 20CM. μg /1gsoil 0
- 26. COLOUR DISTRICT PLACE COLOR SABARKANTHA 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. 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 pH 9 8 7 6 5 10CM,Ph 4 3 20CM,Ph 2 1 0 HIMATNAGAR TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA
- 28. INORGANIC NITROGEN DISTRICT PLACE 10CM, μg/ml 20CM, μg/ml SABARKANTHA 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 3 2.5 2 1.5 1 10CM, μg/ml 20CM, μg/ml 0.5 0
- 29. SULFUR DISTRICT PLACE 10CM, mg/lit 20CM, mg/lt SABARKANTHA 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 SULFUR 120 100 80 60 40 10CM, mg/lit 20 20CM, mg/lt 0
- 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 CARBON 140 120 100 80 60 10CM,mg/lit 40 20CM, mg/lit 20 0
- 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 CHLORIDE 60 50 40 30 20 10CM, mg/100g 20CM, mg/100g 10 0
- 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 CARBONATE 6 5 4 3 2 10CM ,in% 1 20CM ,in% 0
- 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 HARDNESS 4000 3500 3000 2500 2000 1500 10CM, mg/lit 1000 500 20CM, mg/lit 0
- 34. CARBONATE DISTRICT PLACE 10CM,mg/100g 20CM,mg/100g SABARKANTHA 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 CARBONATE 40 35 30 25 20 10CM,mg/100g 15 20CM,mg/100g 10 5 0 HIMATNAGAR TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA
- 35. BICARBONATE DISTRICT PLACE 10CM,mg/100g 20CM,mg/100g SABARKANTHA 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 BICARBONATE 30 25 20 15 10 10CM,mg/100g 5 20CM,mg/100g 0
- 36. CONCLUSION • According tothe 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. 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.