AMIDOHYDROLASES PROFILING FROM SOILS OF NORTH GUJARAT

Guided & Checked By :Dr. S.A. Bhatt sir

Shreya M. ModiMSc Sem-IV

Exam No.- 181

SAMPLING SITES

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

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

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.

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

REQUIREMENT

• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)

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

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

Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O

Mix the content thoroughly

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

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

Make final volume 50ml by addition of KCl-UO2(C2H3O2)2:2H2O

Mix the content thoroughly

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

Add 0.2g MgO

Steam distillate content until 30ml of distillate are collected in the flask

Titrate the distillate with 0.005M H2SO4

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.

Cont….

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

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.

REQUIREMENT

• Apparatus Steam distillation apparatus Incubator adjustable to 37◦C pH meter Volumetric flask (50, 100, 1000, 2000 ml) Automated titration Erlenmeyer flask (100ml)

CHEMICALS

• Toluene• Tham buffer• KCl-Ag2SO4 solution• 0.5M asparagine solution• MgO• 0.005M H2SO4

• Indicator solution

Cont……

• Boric acid indicator solution• 0.05M NaOH• Ammonium standard soluton• 95% ehanol• Distiller water

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 =

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

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

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

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

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

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

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

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

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

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%

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

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

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

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.

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)

Thank You