sumanta_enzymology

7/28/2019 sumanta_enzymology

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EXPERIMENT1

Aim: Isolation of microorganisms from enriched soil.

Principle:

Serial dilution: The method is based upon the principle that when material containing

microorganisms is cultured each viable organism will develop in a colony , serial dilution is done

in order to reduce the number of microorganisms.

Pouring: Only half plate pour for agar media agar was solidified agent help for microbial growth.

Spreading: The spread plate technique is used for the separation of dilution, mixed population of

microorganisms so that individual colony can be obtained. In this technique microorganisms arespread over the solidified agar medium with a sterile L- shape glass rod in such a way when the

petriplate is spun on a turn table single cell will be deposited with the bent glass rod on to the agar

surface. Thus some of the cells will be separated from each other by a distance sufficient to let the

colony to develop, free from each other.

Requirements: .i)0.5 gm soil ,ii) normal saline ,iii) Nutrient Agar media , iv)Test tubes,v) conical

flask ,vi) beaker, vii) petriplates ,viii) Autoclave ,ix) spirit lamp, x) inoculation loop, xi) laminar

air flow, xii) cotton .

Procedure:

0.5 gm of the soil sample was suspended in 5 ml of normal saline mixed thus stocksolution formed.

From the stock solution 0.5 ml solution was pipetted out and poured in the second testtube containing 4.5 ml of normal saline. Now the second test tube mixed.

Each sample was serially diluted and the dilution 10-1 to 10-2. From last three dilution 10-2, 10-3 and 10-4, 500 l solution was pipette out and spreads on

the surface of sterile Petri plates of different prepared solidified agar medium by spread

plate method.

Now the plate were kept for incubation for 4 - 5 days at 37 0C.

Observation: After proper incubation period ; count the numbers of colonies on the plate.


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Result: Different bacterial colonies were seen in the NAM plate. Microbes from soil was

isolated by serial dilution method and mixed culture was obtained. The colonies from these culture

were name as, A1, A2, A3 and A4 having different physical characteristics.

Table 1: Morphological Characteristics Of Colonies

Figure 1: Mixed Culture Plates

S. No. Colony morphology A1 A2 A3 A4

1 Shape Circular Irregular Irregular Spindle

2 Elevation Convex Convex Convex Flat

3 Margin Entire Discrete Entire Curled

4 Pigmentation White Yellow Yellow White

5 Texture Gummy Gummy Gummy Soft

6 Surface Smooth Smooth Smooth Rough

7 Opacity Opaque Opaque Opaque Transparent


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EXPERIMENT2

Aim: Purification of mixed culture by different streaking methods.

Principle: An ample amount of colonies differing in certain features, located on the original agar

plate and physically isolated from all other colonies on the same plate. Generally heterogeneous in

nature, i.e.( that all the degenerate from the different parent). Thus, this can be considered as a

mixed culture.

Requirements: A3 Culture, Petri plate, Spirit lamp, inoculation loop, 70% ethanol, nutrient agar

media, LAF.

Procedure:

NAM was prepared and autoclaved. Poured the media in to the petriplate and let it to solidify in LAF. After solidification, a particular colony was picked by an incinerated inoculating loop and

streaking was done over the media.

Plates were incubated at 370 C for 24 hour in inverted position

Figure 2: Pure Culture Plates

Result: Pure culture was obtained.


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EXPERIMENT3

Aim: Screening of the pure culture obtained from enriched soil for amylase production.

Principle: The screening media is minimal agar media, supplemented with 1% starch. After

inoculation with the interested microorganism and further flooding the plates with iodine solution,

a core zone of inhibition is seen around the amylase positive colonies giving the indication of

utilization of the starch present in the medium by the inoculated cultures.

Requirements : Minimal agar media - supplemented with 1% starch, Iodine,A3 Culture.

Procedure:

Four purified colonies were streaked on the screening media and were kept for incubationfor 2 days. A control plate was also maintained for comparison .

After the 2 days of incubation the plates were flooded with iodine solution in order to checkthe hydrolysis of starch .

Starch hydrolysis was ranked on the basis of visual identification as intense (+++),moderate (++) , slight (+) , no hydrolysis (-) .

Observation:

After addition of the screening plates core zones were observation.

Result:

The hydrolysis activity was quantified based on the visual identification as intense, moderate and

no hydrolysis. The intense depolarization that our colony 1 is amylase enzyme.


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Table 2: Amylase screening.

Result & observation:

Figure 3: Amylase Screeniug

S. No. Culture Character

1 A3 +++

2 A2 ++

3 A1 -

4 A4 +


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:

Figure4:Effect Of pH

Result: Maximum growth was obtained at pH 7.

Effect Of Temperature.Requirements: A3 culture ,4 petri plates , nutrient agar medium , inoculation loop .

Procedure :

Prepare 4 petri plate, streaking A3 culture on solidified agar medium . place 4 different incubation at 28 0C , 22 0C , 50 0C , 37 0C for overnight .

Observation: The maximum growth of the culture A3 was studied and obtained at

370C.

Figure 5: Effect Of Temperature

Result: Maximum growth at temperature 370C.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

pH5 pH7 pH9 pH11

O.D

pH

Effect Of pH


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EXPERIMENT5

Aim: Study the bacterial growth curve.

Principle: Growth may be defined as the orderly increase of all the components of living cells i.e.

its size and its shape, which results from multiplication of cells, bacterial growth is studied by

analyzing the growth curve of a microbial culture . When microorganism are growth in a closed

system or batch culture, the resulting growth curve usually has four phases, the i) lag, ii) log,

iii)stationary and iv) death phase.

Procedure:

100 ml nutrient broth was prepared and autoclaved. 20 ml nutrient broth was taken in flask and made it as blank and 80 ml nutrient broth in a flask

for inoculation.

O. D. was taken after every 24 hrs for 6 day by setting zero with blank.Observation: Growth curve of A3 was studied by taking its absorbance reading at 660 nm at 24

hours interval and the reading as show below were obtained .

Table4: Growth Curve

S. No. Day O. D. at

600nm

1 1 0.04

2 2 0.07

3 3 0.16

4 4 0.18

5 5 0.22

6 6 0.25

7 7 0.25

8 8 0.20

Result: Sigmoid growth curve was obtained.


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EXPERIMENT - 6

Aim: Production of amylase by Submerged fermentation.

Theory: Production of industrially important products by the help of microorganism under

controlled physical and chemical condition in known as fermentation. There are two type of

fomenters are used in industrial level.

a) Flask level: Ordinary normal machine not so much complicated and mainly used in smallindustries. pH ,O2/CO2,Pressure, are need to maintained manually.

b) Shake flask: It is operated manually and pH ,O2/CO2,Pressure, are need to maintainedmanually. Shake flask has to be kept in incubator shaker and it is commercially used in

small industries.

c) Bioreactor/fermentor: Advanced and mainly used in large production industriespH,O2/CO2,pressure,temperature are maintained automatically.

Figure 6 :Bioreactor fermentor

Principle: The proper procedure for a batch fermentation is first to inoculate a small flask of

nutrient broth with pure culture from a petriplate , a culture tube ( containing liquid nutrient ) andthen keep the flask in incubator shaker 72 hrs.

Requirements: Production media, A3 Culture, Shaker, flask.


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Procedure:

Prepare 60 ml production media was prepared and inoculated by A3 liquid culture. Incubated in shaker at 37oC/ 120 rpm for 72 hours ,

Figure 7: Production media of Submerged Fermentation


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EXPERIMENT - 7

Aim: Production of amylase by Solid state fermentation.

Principle: Solid state fermentation has been defined as the fermentation process occurring in the

absence or near absence of free water. solid state fermentation processes generally employ a

natural raw material as carbon and energy source. Wheat bran is the most commonly used

substrate in solid state fermentation.

Requirements: i) Mineral Salt media,

ii) 10 gm wheat bran,

iii) conical flask .

Procedure:

100 ml M. S. media was prepared and moisten 10 gm wheat bran then autoclaved. Then inoculated in 1 ml 24 hour old inoculate culture , media used for 3 days.

Figure 8: Wheat bran moistened by MSM in flask for Solid State Fermentation


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EXPERIMENT -9

Aim: Purification of enzyme (protein) by salt precipitation method.

Principle: Protein solubility is a complex function of the physiochemical nature of the

proteins, pH temperature and the concentration of the salt used. It also depends on whether the

salt is kosomtropic (stabilizes water structure) or chemotropic (disrupts water structure).

Procedure:

Took a beaker containing 50 ml crude enzyme and surrounded it in a containingmaintained ice at 4

0

C.

Placed the whole system on the magnetic stirrer. Poured 23.61 gm ammonium sulphate and pinch by pinch after every 5-7 minute. Stored at refrigerator overnight. Transferred the sample solution in eppendorf tube and spun at 10,000 /10 for 4 0C ,

discarded supernatant and store the pellet .

Took the pellet and 500 l Tris buffer dissolve in pellet and final made up volume 10 ml100 m M Tris buffer , used for dialysis .

Observation : Turbidity is seen during salt precipitation procedure .

Result : Enzyme has been precipitated.


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EXPERIMENT - 10

Aim: To desalt the ammonium sulphate precipitated solution (Dialysis).

Principle: The concentrated protein solution obtained after salt precipitation is placed in the

dialysis bag (semipermeable membrane) with small pores to allow water and salt to pass out to the

bag and protein is retained. Dialysis is done in order to desalt the ammonium sulphate

precipitated solution inside the bag equilibrated with the solution outside with respect to the salt

concentration . Both osmosis and diffusion processes take place in dialysis .

Requirements : 10 ml ( submerged protein ) , Dialysis bag , Thin thread , D/W ,.1% SDS ,100 mM

Tris HCl.

Pretreatment Of bag =>

Procedure:

Dialysis bag boiled for 10 mins in d/w ( 1min x 2 ) ,and boiled for 10 minute in .1% SDS(1min x 2) , then boil for 10 mins in d/w ( 1min x 2) .

Dialysis bag dried by tissue paper and filled in the protein then knoted by thin thread. Dialysis bag placed in 200 ml 100 mM Tris buffer in besaker and buffer change 2 to 3 time

more , stored at 40C for overnight .

Observation: Due to endosmosis bag become tough.

Figure 9: Dialysis Setup

Result: Dialysis has been performed successfully.


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EXPERIMENT11

Aim:

1. To plot standard curve of maltose by DNSA method.

2. Estimation of enzyme activity by DNSA method.

Principle : DNS is 3,5, dinitrosalicylic acid(yellow in color) which reacts with reducing sugar and

gets converted into 3-amino-5nitro salicylic acid(ANSA). DNS acts as stopping agent as well as

coloring agent. It gives brown color.

Reaction: () ().

Enzyme activity =

Requirements: 1% starch in Tris buffer,DNS reagent, Maltose 0.5mg/ml solution,d/w, glasswares

colorimeter, water bath.

Procedure :

To plot standard curve of maltose

10 test tubes were taken along with blank , maltose was poured in all test tubes(exceptblank) in an increase order in such a way that in all test tubes final volume would be 1ml

.

1 ml of DNS was added to all test tubes and were kept at 100 0C in water bath for 15 mins 5 ml d/w was added to all test tubes and vortexed gently. O.Ds were taken @ 540 nm by setting zero with the blank .


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Estimation of enzyme activity

2 test tubes were taken one of them was set as blank. 0.5 ml of crude enzyme was taken in a test tube and 0.5 ml of 1% starch was added to it and

placed in incubator @ 37C for 15 mins.

1ml DNSA was added to both the test tubes and kept them at water bath for 15 mins. 5 ml D/W was added to both the test tubes and vortexed gently. O.D.s were taken @ 540 nm.

Observation:

Table 5: Standard graph for DNS assay

S. No. Volume of

maltose

(.5mg/ml)

D/W Conc. of

maltose

DNS

Boilingfor15m

ins

d/w

Vortexing

O. D. 540 nm

1 0.0 ml 1.0 ml 0 1 ml 5 ml 0.00

2 0.1 ml .9 ml 0.05 1 ml 5 ml 0.04

3 0.2 ml .8 ml 0.1 1 ml 5 ml 0.06

4 0.3 ml .7 ml 0.15 1 ml 5 ml 0.10

5 0.4 ml .6 ml 0.2 1 ml 5 ml 0.13

6 0.5 ml .5 ml 0.25 1 ml 5 ml 0.14

7 0.6 ml .4 ml 0.3 1 ml 5 ml 0.20

8 0.7 ml .3 ml 0.35 1 ml 5 ml 0.23

9 0.8 ml .2 ml 0.4 1 ml 5 ml 0.24

10 0.9 ml .1 ml 0.45 1 ml 5 ml 0.2511 1.0 ml 0.0 ml 0.5 1 ml 5 ml 0.27


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Table 6 :Estimation of enzyme activity by DNS method

Calculation:

Crude enzyme

Batch fermentation:

Enzyme activity= ((.360*1.42)/0.5)/15 U/ml/min

= 0.0681 U/ml/min

Solid State fermentation:

Enzyme activity= ((.360*2.6)/0.5)/15 U/ml/min

=0.1248 U/ml/min

Pure enzyme

Batch fermentation:

Enzyme activity= ((.360*1.06)/.5)/15 U/ml/min

= 0.05088 U/ml/min

SSF: Enzyme activity= ((.360*1.66)/.5)/15 U/ml/min = 0.0796 U/ml/min

Crude enzyme /Pure

Enzyme

(ml)

1%

starch

Incubationat370C

for15mins

DNS

reagent

Boiligfor15minutes

D/W

Vortexing

O. D. 540 nm

Blank _ 0.5 ml 1 ml 5 ml 0.00

Test

(Submerged

fermentation

)

0.5

ml

0.5 ml 1 ml 5 ml 0.77(crude)

0.57(pure)

Test

(Solid State

fermentation

)

0.5

ml

0.5 ml 1ml 5 ml 1.43(crude)

0.87(pure)


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EXPERIMENT12

Aim: To plot standard curve of protein by Bradfords assay.

Principle: The bradfords assay is based on absorbance shift in the dye-coomassie.When the

previously red form coomassie reagent changes and stabilizes into coomassie blue by binding of

protein. The amount of blue complex present in the solution is a measure for the protein

concentration by use of absorbance reading. The bound form of dye has an absorption spectrum

maximum at 595nm.

Reaction:

Requirements:

1. Bradfords reagent : Dissolve 2.5 mg Coomassie Brilliant Blue Dye in 5 ml of 95% Ethanol,

add 5 ml of 85% orthophoshoric acid with constant stirring. Make up the volume to 50 ml

2. Protein Solution:

Stock Standard : 1 BSA : 1 D/W

Working Standard : 1:4

Procedure :

10 test tubes were taken along with blank, BSA was poured in all test tubes(except blank)in an increase order in such a way that in all test tubes final volume would be .5 ml .

2.5 ml reagent BradfordS reagent was added in each test tube vortex and keep the testtubes for 10 minute at room temperature .

O. D.s were taken @ 595 nm and graph between concn. of Protein ( BSA.) vs absorbancewas plotted.


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Table 7: Bradfords Assay

S.

No.

BSA. l D/W l Concentratio

n of BSA.

Bradford

Reagent

ml

Incubationfo

r10minuteindark

V

ortexing

O.D @

660 nm

1 0.0 500 0.0 2.5 ml 0.0

2 50 450 0.02 2.5 ml 0.13

3 100 400 0.03 2.5 ml 0.15

4 150 350 0.05 2.5 ml 0.17

5 200 300 0.07 2.5 ml 0.26

6 250 250 0.08 2.5 ml 0.23

7 300 200 0.1 2.5 ml 0.32

8 350 150 0.12 2.5 ml 0.35

9 400 100 0.14 2.5 ml 0.39

10 450 50 0.15 2.5 ml 0.41

11 500 0 0.17 2.5 ml 0.45


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EXPERIMENT13

Aim:

1. To plot standard curve of protein by Lowrys method.

2. Protein estimation by Lowrys method.

Principle: In this method the color development relies on the formation of copper- protein

complex as in biuret reaction . On the reduction of phosphomolybdate and phosphotungustate

anions present in Folins Cio-calteau phenol reagent by the tyrosine and tryptophan residues of the

proteins , to heteropolymolybdate blue and heteropolytungstate blue respectively , which give a

blue color complex with a max of 660 nm. Cu++ also acts as a catalytic in the reduction reaction .

Reaction:

( )

Blue Colored Complex. max =660nm.

Requirements: BSA , Reagent A , Reagent B , Reagent C, Reagent D , Distilled water , flask ,

pipette ,Commercial Folin Ciocalteau .

Reagent A2% Na2 Co3 in .1N NaOH .

Reagent B -0 .5%CuSo4.5H2O in 1% K Na tartarate.4H2O .

Reagent C50 ml Reagent A + 1ml Reagent B .

Reagent DFolic Ciocalteau Phenol ( 1:1 ratio )

Working BSA 1:4 distilled water.

Procedure :


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To plot standard curve of protein

10 test tubes were taken along with blank, BSA was poured in all test tubes(except blank)in an increase order in such a way that in all test tubes final volume would be 1 ml.

5 ml reagent C was added in each test tubes ,vortexed and kept the test tubes for 10 minsat room temperature .

After 10 mins 0.5 ml reagent D was added in each test tubes and kept the test tubes indark..

O. D.s were taken @ 660 nm and graph between conc. Protein ( BSA ) vs absorbance wasplotted.

Estimation of protein in Crude extract/pure enzyme

2 test tubes were taken and on of them was set as blank. 0.1ml of crude extract /pure enzyme and 0.9 ml of d/w were added in a test tube, then 5 ml

reagent C was poured in the test tube and kept the test tube @ room temperature for 10

mins.

0.5 ml of reagent D was added to the test tube and kept it for 30 mins in dark .whereas inblank 1 ml d/w was added, then 5 ml of reagent C added to it and left tube for 10 mins @

room temperature and finally reagent D was added to the blank and left it for 30 mins in

dark,

O.D.s were taken @ 660 nm

Observation:


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enzyme

(ml)

D/W

(ml)

C

(ml)

D 660 nm

Blank 0.0 1l 5 0.5 ml 0.00

Test

(Submerged

fermentation)

0.1

ml

0.9 5 0.5 ml 0.46(crude)

0.14(pure)

Test(Solid

State

fermentation)

0.1 0.9 5 0.5 1.51(crude)

0.44(pure)

Calculation: Crude extract

1.Amount of protein= 0.295 ml in 1 ml (Submerged fermentation);

For 80 ml enzyme , 23.6 ml of protein.

2. Amount of Protein=0.952 ml in 1 ml(solid state fermentation)

For 15 ml of enzyme, 14.28 ml of protein.

Pure enzyme

3. Amount of protein=0.089 ml in 1 ml ( after dialysis pure enzyme obtained from the crude of

batch fermentation)

For 10 ml enzyme, .89 ml of protein

4. Amount of protein= 0.28 ml in 1 ml (after dialysis/SSF)

For 15 ml of enzyme, 4.2 ml of protein.


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EXPERIMENT14

Aim: To determine the molecular weight of protein (SDSPAGE).

Principle:

SDS is an anionic detergent which bind strongly and denatures protein the number of SDS

molecule bind to a polypeptide chain is approx half the number of amino acid residues in the chain

The protein SDS complex carries net negative charge, hence more towards the anode and the

separation is based on the size of the protein .

Requirements:

Stacking gel , Separating gel , 10%SDS , 30% acrylamide mixture , TEMED , 1M Tris buffer , 1%

ammonium per sulphate , D/W .

Composition:

(A) Gel: Table 10: Reagents

Reagents Separating gel Stacking gel

30% Stock 3.1 ml 0.55 ml

10% SDS 100 l 50 l

1M tris HCl 2.5 ml (pH-8.8) 0.63 ml (6.8)

D/W 3.3 ml 3.5 ml

10 % APS 100 l 50 l

TEMED 15 l 8 l

(B) Sample preparation : 1:1 volume 50 l sample was taken and 50 l buffer .Then incubated at

600C for 5 minute ,then cooled it and loaded .


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Sample buffer : Bromophenol blue .

SDS .

Betamercaptoethanol .

Glycerol / Sucrose .

( C ) Loading and electrophoresis : Movement in prevent of electric buffer

Electric buffer : 196 mM glycine .

0.1% SDS .

50 mM Tris buffer .

(D) Staining solution :.2% C.B.B. R-250 .

Methanol .

Glacial acetic acid .

Distilled water .

(E) Destaining solution :

For 100 ml .

20 Methanol .

5 Glacial Acetic Acid .

25 Distilled water .

Procedure :

Stacking and separating gel were prepared . Continuous system only separating gel 100 % was filled in plate . Discontiuous system separating gel (70%) and stacking gel (30%) was filled in another

plate .

The comb was placed above the running plate and base which contain gel . Sample buffer and electrode buffer were prepared. 3 eppendorf tubes were taken. Equal volume of sample buffer and after dialysis sample1 were taken in 1st eppendorf tube Equal volume of sample buffer and after dialysis sample 2 were taken in 2nd eppendrof tube


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Equal volume of sample buffer and BSA mg/ml were taken in 3rd eppendrof tube . The comb was removed from plate . The running tank was filled with electric buffer . 20 l from the 1st eppendrof tube was taken and poured in the 4th wells. 20 l from the 2nd eppendrof tube was taken and poured in the 7th & 8th wells . 20 l from 3rd eppendrof tube was taken and poured in the 1st ,2nd ,9th ,10th wells. The anode electrode system was connected with the electrode at 55 volt and plate it in the

fridge for 55 mins .

After 30 mins took it out from the fridge and kept it at room temperature at 70 volt for 60mins

The gel was taken out and immersed in staining solution for overnight . After 24 hour the gel was washed with destaining dye .

Figure 10: SDS-PAGE Setup.


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1 2 3 4 5 6 7 8 9 10

1, 2, 9, 10= BSA; 3= crude amylase; 4= pure amylase; 5= crude amylase ssf; 7, 8= pure amylase (ssf)

Figure11:Destained PAGE under White Ligh Transilluminator.

Observation : Blue colour band was observed .

Result : The molecule weight of amylase isolated is approximately 66 Kda .


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EXPERIMENT15

Aim: Characterization of pure enzyme(effect of temperature , pH , activator , inhibitor ).

Requirements : DNS reagent , test tube , NaOH , HCl , Mg2+

, Ca2+

, SDS , EDTA , distilled water

Procedure:

For temperature :

5 test tubes were taken label on blank . Blank was set at 1st test tube . .5 ml enzyme was taken in 2nd test tube and .5 ml 1% starch was added and incubated at 20

0C for 15 minute and 1 ml DNS reagent was added and boiled then 5 ml distilled water was

added and O. D.was taken .

.5 ml enzyme was taken in 3rd test tube and .5 ml 1% starch was added and incubated at 370C for 15 minute and 1 ml DNS reagent was added and boiled then 5 ml distilled water was


0.5 ml enzyme was taken in 4th test tube and .5 ml 1% starch was added and incubated at 200C for 15 minute and 1 ml DNS reagent was added and boiled then 5 ml distilled water was


0.5 ml enzyme was taken in 5th test tube and .5 ml 1% starch was added and incubated atroom temperature for 15 minute and 1 ml DNS reagent was added and boiled then 5 ml

distilled water was added and O. D. was taken .

Table11: Effect Of Temperature on enzyme

Temperature O.D.

22c 0.88

Room temperature 0.87

37c 0.86

50c 0.96


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Figure 12:Effect of temperature

For pH :

5 test tubes were taken label on blank . Blank was set at 1st test tube . 0.5 ml enzyme was added in 2nd test tube and .5 ml 1 % starch was added and maintained

pH5 incubated at 370C for 15 minutes added 1 ml DNS reagent and boiled then added 5

ml distilled water and O. D was taken .

0.5 ml enzyme was added in 3rd test tube and .5 ml 1 % starch was added and maintainedpH7 incubated at 37

0C for 15 minutes added 1 ml DNS reagent and boiled then added 5


0.5 ml enzyme was added in 4th test tube and .5 ml 1 % starch was added and maintainedpH9 incubated at 37

0C for 15 minutes added 1 ml DNS reagent and boiled then added 5


0.5 ml enzyme was added in 5th test tube and .5 ml 1 % starch was added and maintainedpH11 incubated at 37

0C for 15 minutes added 1 ml DNS reagent and boiled then added

5 ml distilled water and O. D was taken .

0.8

0.82

0.84

0.86

0.88

0.9

0.920.94

0.96

0.98

22c 28c 37c 50c

O.D.

Temperature

Effect of temperature

O.D


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Table 12: Effect Of pH on enzyme

pH O.D.

pH 5 0.56

pH 7 0.78

pH 9 .62

pH 11 0.68

Figure 13: Effect of pH

For Activator:

3 test tubes were taken on blank Blank was set without activator . 0.5 ml enzyme was taken in 2nd test tube and 0.5 ml 1% starch and 0.2 Ca2+ was

added to it and incubated at 370C for 15 minute then 1 ml DNS was added and

boiled then 5 ml distilled water was and O. D. was taken.

0.5 ml enzyme was taken in 3rd test tube and 0.5 ml 1% starch and 0.2 Mg2+incubated at 37

0C for 15 minute and added 1 ml DNS and boil it then 5 ml distilled

water was added and O. D was taken.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

pH 5 pH 7 pH 9 pH 11

O.D.

pH

Effect of pH

O.D.


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Table 13: Effect of activator on enzyme.

Activator O.D

Mg+

0.54

Ca + 0.60

Figure 14:Effect of activator.

For inhibitor :

3 test tubes were taken on blank. Blank was set without activator . 0.5 ml enzyme was taken in 2nd test tube and 0.5 ml 1% starch and 0.2 SDS was

add and incubated at 370C for 15 minute then 1 ml DNS was added and boiled then

5 ml distilled water was added and O. D. was taken.

0.5 ml enzyme was taken in 3rd test tube and 0.5 ml 1% starch and 0.2 EDTAincubated it at 37

0C for 15 minute and added 1 ml DNS and boiled then added 5 ml

distilled water then O. D. was taken.

0.51

0.52

0.53

0.54

0.55

0.56

0.57

0.58

0.59

0.6

0.61

Mg2+ Ca2+

O.D.

Activetor

Effect of activator.

O.D


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Table 14: Effect of inhibitor on enzyme

Inhibitor O.D.

EDTA 0.52

SDS 0.42

Figure15: Effect Of Inhibitor

Result: The enzyme was stable all through the experiment.

0

0.1

0.2

0.3

0.4

0.5

0.6

EDTA SDS

O.D.

Inhibitor

Effect of Inhibitor

O.D.


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APPENDIX

NUTRIENT AGAR MEDIUM (NAM)

S. No. Ingredients Quantity

1 Beef extract 3.0 gm

2 Peptone 5.0 gm

3 Nacl 5.0 gm

4 Agar 15.0 gm

5 Distilled water 1000 ml

(pH-6.8)

NUTRIENT-BROTH (NB)

S. No. Ingredients Quantity

1 Beef extract 3.0 gm

2 Peptone 5.0 gm

3 Nacl 5.0 gm


(pH-6.8)

NORMAL SALINE (NS)

S. No. Ingredients Quantity1 Nacl 8.0 gm


Minimal Agar Media (MAM)

S.No Ingredient Quantity

1 Disodium hydrogenphosphate

6.0 gm

2 Sodium di hydigen phosphate 3.0 gm

3 Nacl 5.0 gm

4 NH4cl 2.0 gm

5 Mg(So4)2 0.1Gm6 Glucose 8 gm

7 Kcl 0.1 gm

8 Agar 15 gm

9 Distilled water 1 lit

( pH 7 )


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DNS Reagent:

10 gm of DNS Powder dissolved in 500 ml of distilled water. 300 gm of K NA Tartarate was added. 200 ml of 2 N NaOH was added And later made up the volume up to 1000 ml by adding distilled water.

Production Media :

S. No Ingredient Quantity

1 Peptone 5gm

2 Yest Extract 5 gm

3 NaH2Po4 1 gm

4 MgSO4 0.2 gm

5 CaCo3 0.2 gm

6 KCl 0.2 gm

7 NaCl 5 gm

8 Starch 7 gm

9 Distilled Water 1000 ml

( pH 7 )

Mineral Salt Media (MSM):

S. No Ingredient Quantity

1 NaH2PO4 2 gm

2 Na2HPO4 6 gm

3 NaCl 5 gm

4 NH4Cl 2gm

5 MgSO4 0.1 gm

6 C6H12O6 8 gm

7 KCl 1 gm

8 Distilled Water 100 ml

(pH 7 )


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sumanta_enzymology

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