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AMIDOHYDROLASES PROFILING FROM SOILS OF NORTH GUJARAT
Guided & Checked By :Dr. S.A. Bhatt sir
Shreya M. ModiMSc Sem-IV
Exam No.- 181
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SAMPLING SITES
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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
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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
NH3 + RCOOHAmidaseRCONH2 + H2O
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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.
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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
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REQUIREMENT
• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)
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PROCEDURE
Take 50ml volumetric flask
Add 5g moist soil Add 0.2ml Toluene and 9ml Tris buffer
Mix it well
Add 1 ml 0.5M Amide solution
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Mix well for few 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
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Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O
Mix the content thoroughly
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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
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Incubate for 2 hrs 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
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Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O
Mix the content thoroughly
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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
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Add 0.2g MgO
Steam distillate content until 30ml of distillate are collected in the flask
Titrate the distillate with 0.005M H2SO4
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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.
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Cont….
• It catalyses the hydrolysis of L-aspargine, which produce L-aspartic acid and ammonia.
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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.
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REQUIREMENT
• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)
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CHEMICALS
• Toluene• Tham buffer• KCl-Ag2SO4 solution• 0.5M asparagine solution• MgO• 0.005M H2SO4
• Indicator solution
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Cont……
• Boric acid indicator solution• 0.05M NaOH• Ammonium standard soluton• 95% ehanol• Distiller water
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CALCULATION
C × 50 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
L-asparginase activity =
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PHYSICOCHEMICAL ANALYSISNo 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
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AMIDASE PLACE 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
HIMATN
AGAR
TALO
D
MODASA
VIJAPUR
VISNAGAR
TARABH
NEDARA
CHANASMA
ADIYA0
100200300400500600700800900
1000AMIDASE
1OCM. μg /1gsoil20CM. μg /1gsoil
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L-ASPARGINASE
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
100
200
300
400
500
600
L-ASPARGINASE
1OCM. μg /1gsoil20CM. μg /1gsoil
PLACE 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
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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
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pH DISTRICT PLACE 10cm,pH 20cm,pH
SABARKANTHA
HIMATNAGAR 8.5 7.5TALOD 7 7.5
MODASA 7 7
MEHSANAVIJAPUR 7.5 7.5
VISNAGAR 8 8TARABH 7.5 8
PATAN
NEDARA 8 7.5CHANASMA 8 7.5
ADIYA 8 7.5
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0123456789
pH
10CM,Ph20CM,Ph
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HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
0.5
1
1.5
2
2.5
3
INORGANIC NITROGEN
10CM, μg/ml20CM, μg/ml
INORGANIC NITROGENDISTRICT PLACE 10CM, μg/ml 20CM, μg/ml
SABARKANTHA HIMATNAGAR 0.222 0.825TALOD 0.412 0.349
MODASA 0.38 0.38MEHSANA VIJAPUR 1.174 0.73
VISNAGAR 0.38 0.666TARABH 0.317 0.158
PATAN NEDARA 1.17 0.73CHANASMA 1.71 1.96
ADIYA 2.53 2.15
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SULFUR DISTRICT PLACE 10CM, mg/lit 20CM, mg/lt
SABARKANTHA HIMATNAGAR 40 60TALOD 86.66 100
MODASA 73.33 46.66MEHSANA VIJAPUR 73.33 53.33
VISNAGAR 86.66 60TARABH 53.33 53.33
PATAN NEDARA 40 53.33CHANASMA 41.76 38.51
ADIYA 52.27 46.13
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
20
40
60
80
100
120
SULFUR
10CM, mg/lit20CM, mg/lt
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CARBONDISTRICT PLACE 10CM,mg/lit, 20CM, mg/lit,
SABARKANTHA HIMATNAGAR 125.2 107.8TALOD 94.6 109.4
MODASA 113.8 114.6MEHSANA VIJAPUR 104.6 107.4
VISNAGAR 95 106.8TARABH 125.8 131.8
PATAN NEDARA 112.2 102.8CHANASMA 133.2 118.6
ADIYA 113.6 131
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
20
40
60
80
100
120
140
CARBON
10CM,mg/lit20CM, mg/lit
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CHLORIDE DISTRICT PLACE 10CM, mg/100g 20CM,
mg/100gSABARKANTHA HIMATNAGAR 39.4 55.38
TALOD 31.95 35.5MODASA 24.49 31.95
MEHSANA VIJAPUR 24.85 46.15VISNAGAR 31.95 29.11
TARABH 56.8 35.14PATAN NEDARA 56.09 39.05
CHANASMA 49.7 39.76ADIYA 42.6 35.5
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
10
20
30
40
50
60
CHLORIDE
10CM, mg/100g20CM, mg/100g
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CALCIUM CARBONATE DISTRICT PLACE 10CM ,in% 20CM ,in%
SABARKANTHA HIMATNAGAR 3.5 3TALOD 2.5 2.5
MODASA 3 2MEHSANA VIJAPUR 1 0.5
VISNAGAR 5 5.5TARABH 2 1.5
PATAN NEDARA 3 3CHANASMA 4.5 5.5
ADIYA 3.5 4
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
1
2
3
4
5
6
CALCIUM CARBONATE
10CM ,in%20CM ,in%
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TOTAL HARDNESSDISTRICT PLACE 10CM, mg/lit 20CM, mg/lit
SABARKANTHA HIMATNAGAR 940 920TALOD 700 660
MODASA 700 660MEHSANA VIJAPUR 280 800
VISNAGAR 940 800TARABH 800 1300
PATAN NEDARA 640 700CHANASMA 1000 3500
ADIYA 700 1240
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
500
1000
1500
2000
2500
3000
3500
4000
TOTAL HARDNESS
10CM, mg/lit20CM, mg/lit
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CARBONATEDISTRICT PLACE 10CM,mg/100g 20CM,mg/100g
SABARKANTHA HIMATNAGAR 12 10.5TALOD 9.3 7.5
MODASA 10.5 7.5MEHSANA VIJAPUR 10.5 6
VISNAGAR 12 9TARABH 35.1 12
PATAN NEDARA 7.5 9CHANASMA 6.3 12
ADIYA 6 7.5
HIMATNAGAR TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
5
10
15
20
25
30
35
40
CARBONATE
10CM,mg/100g20CM,mg/100g
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BICARBONATEDISTRICT PLACE 10CM,mg/100g 20CM,mg/100g
SABARKANTHA HIMATNAGAR 12.2 9.15TALOD 6.1 9.15
MODASA 18.3 9.15MEHSANA VIJAPUR 24.4 9.15
VISNAGAR 12.2 12.2TARABH 18.3 15.25
PATAN NEDARA 12.2 21.35CHANASMA 15.25 10.67
ADIYA 24.4 21.35
HIMAT-NAGAR
TALOD MODASA VIJAPUR VISNAGAR TARABH NEDARA CHANASMA ADIYA0
5
10
15
20
25
30
BICARBONATE
10CM,mg/100g20CM,mg/100g
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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.
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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)
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Thank You