1. AMIDOHYDROLASES PROFILING
FROM SOILS OF NORTH GUJARAT
Guided & Checked By :
Dr. S.A. Bhatt sir
Shreya M. Modi
MSc Sem-IV
Exam No.- 181

2. SAMPLING SITES

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. 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. 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. 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. 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 volumetric flask
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 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

10. Make final volume 50ml 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 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

13. Make final volume 50ml by addition of
KCl-UO2(C2H3O2)2:2H2O
Mix the content thoroughly

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. 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 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. Cont….
• It catalyses the hydrolysis of L-aspargine,
which produce L-aspartic acid and ammonia.

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. 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 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. 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. Thank You