Detection of alpha amylase and protease from soil bacteria through qualitative and quantitative analysis has been done.

1. DISSERTATION
ON
OPTIMIZATION OF CULTURE CONDITION AND
DETECTION OF AMYLASE AND PROTEASE
ENZYME FROM SOIL BACTERIA
Submitted by-
Shaifali Bhargava,
B.Sc. Biotechnology
3rd year
(2016-2017)
Submitted to :-
Dr Ranjana Agarwal
(HOD)
Kanoria PG Mahila Mahavidyalaya, Jaipur
University of Rajasthan

2. INTRODUCTION
Soil consists of several bacteria's which have the capability to produce enzymes
like amylase, protease etc which act as a catalyst to bring about a specific
biochemical reaction.
1) Amylase 2) Protease
Starch hydrolysis sugars Proteins proteolysis peptones + peptides
performed in Starch agar media performed in gelatin agar media

3. Materials-
STUDY SITE AND SAMPLE COLLECTION
Five soil samples were collected in clean dry sterile containers from different soil
environments as follows: From Jagatpura to Nahargarh.
Preparation of Media:-
1) Starch Agar Media – for alpha amylase production
2) Gelatin Agar Media – for protease production
3) Luria Broth - for the detection of amylase activity

4. About 39ml of dihydrogen sodium phosphate was mixed with 61ml of disodium
hydrogen phosphate
To make up 100ml of solution, now pH becomes 7
**For pH 6 and 8 concentration of reagents (5·3:94·7) and (87·3:12·3) was taken
3) ·2% of Starch solution (0·1 and 0·5% solution are also prepared)
Procedure- 0·2g in 100ml of distilled water
Reagents used:-
1) Monobasic sodium phosphate (200mM):- 2·78g of sodium dihydrogen
phosphate was dissolved in 100ml distilled water.
2) Dibasic sodium phosphate (200mM) :- 3·4g of disodium hydrogen
phosphate was dissolved in 100ml distilled water.
Procedure-
Fig: showing prepared phosphate buffer
(conc. 61ml and 39 ml for pH 7), starch solution (0.2%)
Preparation of Buffers and solutions

5. 4) DNS Reagent (Dinitrosalicylic acid)
Procedure-
1g of of dinitrosalicylic acid (DNS) was dissolved in 50ml of distilled water
To the solution, add 39g of sodium tartarate terahydrate in small lots, the solution
turned yellow in colour
20ml of 2N NaOH was added, which turned solution to transparent
orange-yellow colour
The final volume was made to 100ml with distilled water
The solution was stored in amber coloured
bottle.

6. Methods:-
AIM:- OPTIMIZATION OF CULTURE CONDITION AND DETECTION OF
ALPHA AMYLASE AND PROTEASE FROM SOIL BACTERIA
 Starch Hydrolysis test
 Qualitative test
 Amylase Assay
 Determination of amylase activity
 Effect of environmental conditions on enzyme
activity
 DNS Method (Quantitative test)
 Protease Assay

7. 1) Starch Hydrolysis Test
Objective:- For screening alpha amylase producing bacteria
Procedure:-
Prepare Starch agar media
Autoclave for 15 minutes
Media transfer to petri plates and allow to solidy
Pure culture of serial dilution of 10-5, 10-6and 10-7 Streaked on Starch Agar plate
Incubate at 37ºC for 24 to 43 hours
Note the observation
After incubation, 1% iodine solution was
flooded on the starch plate

8. Observation:-
Result :- Clear zones of hydrolysis were considered as amylase producers.

9. 2) QUALITATIVE TEST
It is an auxiliary technique used to enhance the image of micro-organism viewed
under microscope. Stains or dyes are colorful (generally) agents which provide color
to particles under microscope and highlight their structures of biological tissues or
cells or organelles.

10. a) Gram’s staining
It is a kind of differential staining
which differentiate bacteria on the basis
of difference in cell wall of gram
negative and gram positive bacteria.
Observation-
Fig showing Sample 1 is Gram positive
bacteria
Procedure –

11. S.no Samples Observations Gram’s staining reaction
1 Sample 1 Rod shaped,purple colour Gram +ve
2 Sample 2 Rod shaped, purple Gram +ve
3 Sample 3 Pink Gram -ve
4 Sample 4 Purple Gram +ve
5 Sample 5 Purple Gram +ve
Result and interpretation-
2) Negative Staining-
Capsules are composed primarily of
polysaccharides or glycoprotein and destroyed
by heating and hence direct staining methods
cannot be utilized and Nigrosine dye is used to
visualize the capsule
Procedure –

12. Observation-
Fig showing sample 3 contains ecapsulated bacteria
Sno Sample Observation Negative staining reaction
1 Sample1 Sparkling background
and dark bacteria
Encapsulated
2 Sample2 Sparkling background and
dark bacteria
Encapsulated
3 Sample3 Dark background with
sparkling bacteria
Ecapsulated
4 Sample4 Sparkling background and
dark bacteria
Encapsulated
5 Sample5 Sparkling background and
dark bacteria
Encapsulated
Result-

13. Result-
3) Simmon Citrate Utilization Test
 Citrate utilization test is used to determine
the ability of bacteria to utilize sodium
citrate as its only carbon source.
 Bromothymol blue indicator is used, turning
from green to blue the color.
Streak the inoculum on
the citrate agar slants.
Bromothymol blue
indicator is used, turning
from green to blue
Sno Sample Observation MR test
1 Sample1 Red positive
2 Sample2 Red positive
3 Sample3 Red positive
4 Sample4 Red positive
5 Sample5 Red Positive
Procedure-
Positive test = If the colour of agar slant
changes to blue.
Negative test = If no change in colour

14. Observation-
Fig showing Agar slants of simmon citrate remains green in
color in all five sample
Sno Sample Observation Simmon citrate test
1 Sample1 Green +ve
2 Sample2 Green +ve
3 Sample3 Green +ve
4 Sample4 Green +ve
5 Sample5 Green +ve
Result-

15. Observation-
6) Catalase Test-
 The Catalase test involves adding hydrogen
peroxide to a culture sample or agar slant.
 If the bacteria produce Catalase, they will
convert the hydrogen peroxide into water
and oxygen gas. The evolution of gas,
causing bubbles, is indicative of a positive
test.
Fig: Effervescence or bubbles indicating the presence
of catalase
Take 200µl of hydrogen peroxide
and inoculate
with loopful of culture.
Effervescence or bubbles
indicate
presence of catalase.
Procedure-

16. Sno Sample Observation Catalase test
1 Sample1 Bubble formation +ve
2 Sample2 No efferences -ve
3 Sample3 Bubble formation +ve
4 Sample4 No efferences -ve
5 Sample5 No efferences -ve
Result-
We concluded that the bacteria which produces a large no. of
enzymes like amylase is Bacillus, gram positive, encapsulated.
Conclusion

17. 3) Amylase Assay
Determination
of amylase
activity
Effect of
environmental
conditions
pH Substrate

18. I) Determination of Amylase activity
0.5 ml
buffer
0.5 ml
starch
2.5ml water
BLANK
Observe O.D at 620 nm
2.5ml of water
500µl of Gram’s Iodine
500µl of starch solution (0.2%) and mixing in
vortex
500µl of phosphate buffer (pH=7)
50µl of sample (lunia broth is prepared and
inoculated)
TEST

19. Sample Colonies Serial dilution O.D (620nm)
S1 C1 10-6 0.0374
S1 C2 10-6 0.4804
S1 C3 10-5 0.5234
S1 C4 10-4 0.5685
S2 C5 10-5 0.1282
S2 C6 10-6 -0.3416
S2 C7 10-4 -0.3591
S3 C8 10-5 0.0138
S3 C9 10-6 0.1282
S3 C10 10-4 0.0123
S4 C11 10-5 -0.0311
S4 C12 10-7 -0.2677
S4 C13 10-6 0.2065
S5 C14 10-6 0.1800
S5 C15 10-5 0.1710
S5 C16 10-4 0.1683
Determination of amylase activity (Blank- 0.00)

20. 0.4804
0.5234
0.5685 0.6
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
S1 10-6 S1 10-5 S1 10-4 estimated
Effect of Serial dilution
It shows that serial dilution effect the production of amylase enzyme

21. II) The Effect of Environment conditions on Enzyme
activity
Effect of pH
Effect of substrate
concentrations
In part I, the buffer that was added – pH-
7
To check activity 2 buffer added with
different pH -6 and 8
To prepare this certain ratio has to
adjusted-NaH2PO4: Na2HPO4
For pH 7 39:61
For pH 6 87.7:12.3
For pH 8 5.3:94.7
In part I, the concentration of
starch was taken 0.2% for
detecting the effect concentration
0.1%and 0.5% solution added

22. Effect of pH
Sample Colonies Serial
dilution
OD at 620nm for
pH-6
OD at 620nm for
pH 8
S1 C1 10-6 0.03987 0.0118
S1 C2 10-6 0.4040 0.4326
S1 C3 10-5 0.0191 0.4318
S1 C4 10-4 0.0050 0.4318
S2 C5 10-5 0.0198 0.0056
S2 C6 10-6 -0.0198 0.0260
S2 C7 10-4 -0.0217 -0.4106
S3 C8 10-5 0.0234 0.0022
S3 C9 10-6 0.0098 0.0138
S3 C10 10-4 0.0090 0.4317
S4 C11 10-5 -0.0116 -0.4103
S4 C12 10-7 0.0488 -0.4217
S4 C13 10-6 0.0055 -0.2931
S5 C14 10-6 0.0205 0.0678
S5 C15 10-5 0.1088 0.0734
4

23. Effect of substrate concentration
Sample Colonies Serial
dilution
OD at 620nm for 0.1%
starch solution
OD at 620nm for 0.5%
starch solution
S1 C1 10-6 0.0228 0.4864
S1 C2 10-6 0.0221 0.4889
S1 C3 10-5 0.4408 0.4858
S1 C4 10-4 0.0200 0.0158
S2 C5 10-5 0.0141 0.0187
S2 C6 10-6 0.0130 -0.4015
S2 C7 10-4 0.0217 -0.0105
S3 C8 10-5 -0.3474 0.0348
S3 C9 10-6 -0.3476 0.0276
S3 C10 10-4 -0.3476 0.0213
S4 C11 10-5 0.0434 -0.4381
S4 C12 10-7 -0.0083 -0.0713
S4 C13 10-6 -0.0016 0.4359
S5 C14 10-6 0.0070 -0.0439
S5 C15 10-5 0.1379 -0.0284

24. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
starch
0.1%
starch0.2% starch
0.5%
estimated
Concentration of amylase activity
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
pH6 pH7 pH8 estimated
Concentration of amylase activity
It shows that pH and starch concentration adversely effect the
production of amylase, it could speed up the rate of reaction or
denature it

25. Fig: Purple color shows the positive
result Indicating the production of amylase
Fig; showing microtubes after
centrifuge of all 16 colonies
Fig: showing purple colour indicates the
presence of amylase in cuvette
Results-
Fig: showing test tubes containing starch
solution of 0.1%

26. Procedure-
Blank:-
Mix 250µl of starch solution with 500µl of
DNS reagents
Boil the solution in water bath for 5
minutes
Cool the solution in a running tap water
Add 1ml of distilled water
4) DNS Method (Quantitative test)
DNS (Dinitro Salicylic acid) – By adding this, the reaction stops and it will give
the exact concentration of the amylase produced.
The bacterial species that showed positive response to alpha-amylase activity,
the samples were subjected to activity assay via DNS method. The bacterial were
grown in LB medium overnight at 37ºC.

27. Test:-
Note O.D at 520nm in spectrophotometer
Add 1ml of distilled water in the solution
Cool the solution in a running tap water
Boil the solution in a water bath for 5 minute
Add 500µl of DNS reagents
Add 250µl of starch solution to the mixture
Add 250µl of the supernatant in micro tubes and discard the pellet
The samples were centrifuged for 10 minutes at 10,000 RPM

28. Observation-
Fig: showing dark colour
after adding DNS reagent
Sno Sample Colonies Serial dilution Concentration of
amylase
estimated
1 S1 C1 10-6 -0.3431
2 S1 C2 10-6 -03532
3 S1 C3 10-5 0.0298
4 S1 C4 10-4 0.1126
5 S2 C5 10-5 0.0409
6 S2 C6 10-6 -0.3598
7 S2 C7 10-4 -0.1420
8 S3 C8 10-5 0.0428
9 S3 C9 10-6 0.4505
10 S3 C10 10-4 0.4588
11 S4 C11 10-5 -0.3923
12 S4 C12 10-7 -0.3130
13 S4 C13 10-6 -0.2621
14 S5 C14 10-6 -0.3016
15 S5 C15 10-5 -0.0009
16 S5 C16 10-6 -0.2831
Result-

29. 4) Protease Assay
Detection of protease enzyme-
Procedure-
Inoculate the culture in deep tubes containing media
Prepare Gelatin Agar media
Observation-
Incubate it at 37ºC for 24 hours
Note the Observation
Fig : Liquification of gelatin agar media shows proteolysis of
protein by protease enzyme

30. Conclusion
 Various bacterial isolates from soil were studied for amylase and protease
producing activity. Amylase and proteolytic activity was measured for high
enzyme producing strain Structural, staining and biochemical activity results
have revealed it as a Bacillus species.
 Status of starch, the amylase concentration and the optimum conditions for
amylase action are also the factors that are influencing the rate of starch
hydrolysis.
 Alpha-amylase activity can be detected from different species of soil
dwelling microorganisms particularly bacteria.
 The majority of the soil bacteria that show alpha-amylase activity belong to
the genus Bacillus, they belong to the family Bacillaceae that are Gram-
positive, spore-forming, aerobic or facultative anaerobe and rod in shape.
 The variation in the pH and the temperature can be attributed to the type of
bacteria and the soil type.

31. Applications
 Amylase is used in industry sector.
 The first enzyme produced industrially was an amylase from fungal source
reported in 1894, which was used as a pharmaceutical aid for the treatment of
digestive disorder.
 It is used in brewing and fermentation industries for the conversion of starch to
fermentable sugar.
 It is used in the textile industry for designing textiles
 It is used to make the mixture with protease and lipase to laundry clothes.
 It is used in the paper industry for sizing.
 It is used in the food industry for the preparation of sweet syrups.
 It is used in jelly industries for the removal of starch in jelly production.