This document summarizes the steps taken to partition and distill the therapeutic enzyme nattokinase from bacteria. Key steps included isolating nattokinase-producing bacteria from soil samples, optimizing growth media, conducting fermentation and downstream processing to extract the crude enzyme, and using HPLC to purify and identify the enzyme. Graphs are presented showing the effects of varying media components and conditions on bacterial growth and nattokinase concentration.

1. Partitioning and Distillation of Therapeutic Enzyme
“Nattokinase”
By:
Sumit Singh
Biotechnology Engineer

2. OBJECTIVE
1. Sample Collection
2. Isolation of Bacteria
3. Screening of Nattokinase producing Bacteria.
4. Media Selection and optimization.
5. Fermentation and Downstream Processing.
6. Estimation of Nattokinase.
7. Strain Identification.
8. HPLC

3. Sample Collection, Serial Dilution
* Collecting the soil sample from the Butcher house.
* Serial diluting the sample by using NaCl to get a desired concentration. We put NaCl because to
make it Isotonic so that the cell would not get burst.
* Preparing NAM media for pouring the diluted sample on it. We prepare the NAM by mixing by
adding Nutrient Agar 2.1gm and Agar-Agar 0.375gm in 45ml distilled water.
* After making the media we sterilize by putting it in the Autoclave for 30min at 121psi.
* Then we pour the media in the Petri plates which contain 15ml each and leave it in Laminar Air Flow
to get solidify.
* After pouring the sample over the NAM media and leaving for 1 night we see growth of colonies on
the media and after that we count the number of colonies and do streaking.

4. * Preparing NAM media and Milk powder in separate flask and mixing them after sterilizing
them by putting them in the Autoclave for 30min.
* Pouring the media on the petri plates and leave them to get dried up. We leave the media for
15 to 20min to get solidified in the Laminar Air flow.
* Now after solidification we draw a line 2.5cm from the sides and do the streaking between
them with the help of scale and we do streaking in between those lines.
* Now after streaking out of 13 petri plates 12 are going to be placed at 37 ℃ And 1 petri plate
is the Control one which we going to be placed at 4 ℃ So we placed that we placed that 1
particular plate in the fridge and rest in the Incubator.
* Leave it for 1 night to check the results and pour Bradford reagent . We select the best result
among them by checking which one has better transparency.
* We select that particular plate for further use.

5. * Here we use the Media Selection process to check the best growth of the Bacteria.
* We use certain compounds like Dextrose, Maltose, Yeast, Peptone, K2
HPo4
, Nah2
Po4
, MgSo4
,
CaCl2
, NaCl, Nh4
Cl, etc. and put them in the test tubes in calculated amount.
* We make 10ml mixture of all these compound and transfer into 2 test tubes in equal amount and
mark it PM2
and PM2
B.
* Then we place both the test tubes PM2
and PM2
B and put in Autoclave for sterilization.
* And then we dip the culture in it with the help of Inoculum and them we place the blank one in
the fridge and the non blank one in the Incubator.
* And then we leave both for 48 hours and after that we take the OD to check the growth. We take
the OD with the help of Spectrophotometer by placing the wavelength at 620nm.
* Those with maximum value show the maximum growth. We use only PM2 ones because it has
maximum value
Media Selection and Optimization

6. Media Optimization uisng Sucrose, Maltose, Dextrose
* Here we optimize media to check the growth of the bacteria. Some of them are Peptone, Yeast,
K2
HPo4
, MgSo4
, CaCl2
. And now we are going to add all these components to check the
growth of our culture by taking OD.
* We mix all this components in flask in 30ml of water and then leave it for sterilization in the
Autoclave at fixed temperature and pressure.
* After then we put all our components in all the 3 pairs of test tubes in equal amounts which
also contain Sucrose, Maltose and Dextrose in their respective test tubes and mark them so
that we can differentiate the blank ones from the non blanks.
* Then after sterilization we put the test tubes in the laminar air flow. After that we pick our
culture with the inoculation loop and dip into all those test tubes.
* Then we place all the blank ones in the fridge at 4 degree and the non blank ones in the
Incubator shaker for up to 48 hours.
* After 48 hours we take the OD.

7. Media Optimization using Yeast and Peptone
* Here we again optimize the media by using Sucrose, K2
HPo4
, MgSo4
, CaCl2
as our key
components along with NAM.
* We make it for 30ml of water mix it in specified amounts and transfer to different test tubes and
marked them.
* Now after that we pour that media in 3 pairs of test tube.
* After that we put measured amounts of Yeast, Peptone and Yeast + peptone in the pairs. Then
after we put all our test tubes in Autoclave for sterilization.
* After that we place our sterilize test tubes in laminar air flow then pick our culture with the help
of Inoculation loop and dip the culture in all the pairs and put the blanks one in the fridge and
the other non blanks one in Incubator.
* After 44 hours we take OD and measure the growth.

8. Media and pH optimizing using MgSo4
, CuSo4
and ZnSo4
* Here we again optimize the media but this time we add Sucrose, K2
HPo4,
CaCl2
, Yeast for 30ml
of distilled water.
* Then we add all our component in test tubes and add MgSo4
, ZnSo4
, CuSo4
in different
respective test tubes and marked it for identification.
* After that we separately optimize the pH too to check the growth with same component in the
previous one.
* Through the same process we take 3 pairs for media check and 5 different tubes to check the
growth of Bacteria in different pH. We mark the test tubes like pH4 , pH9, pH7, pH11, and one
blank.
* Now we inoculate all the test tubes with the culture and put the blank one in fridge and non
blank one in Shaker.
* After 44 hours we take OD to check the Growth. Here we check the growth both in pH and the
other one.

9. Media Optimization using NaCl, CaCl2
, Mgcl2
* So here we optimize the media to check the growth. The composition is Sucrose, Yeast,
K2
HPo4
, MgSo4
.
* We have prepared it for 30 ml of water and then pour it into 3 pairs of test tubes and after that
we add NaCl, CaCl2
, Mgcl2
in their respective tubes.
* After that we sterilize them by placing them in Autoclave and after that we place them in
Laminar Air Flow so to get Aseptic conditions.
* After that we inoculate them in the laminar air flow with the help of Inoculation loop and dip in
the test tubes except the blank one and leave them for 44 hours, one in fridge and the other one
in Shaker.
* After 44 hours we take the OD and check the growth quantity in each test tubes with the help of
Spectrophotometer.
* The tube which give the best results is noted along with the component present inside it. So
through this way we optimize the media.

10. Media Optimization using K2
hPo4
, Kh2
Po4,
NaH2
Po4
* Here we Optimize the media by adding Sucrose, NaCl, Yeast, MgSo4
.
* We prepare this for 3 pairs of test tubes as we are doing before and add K2
HPo4, NaH2
Po4
,
Kh2
Po4
in different pairs of test tube and marked it.
* And we sterilize them by placing them in Autoclave and after sterilization we put all those
test tubes in Laminar Air Flow where we inoculate the culture in all the 3 pairs.
* We inoculate our culture by picking our colonies with the help of Inoculation loop(we should
remember to heat sterilize the loop before picking the colonies). Then we put the blank one in
the fridge at 4 ℃ and the non blank one in Shaker.
* After 44 hours we measure the growth quantity with the help with Spectrophotometer.
* This one is the final media optimization for our culture. Here our Media Selection is over and
throughout this process we come to know which specific component is better for growth for
Bacteria Subtillis.

11. Strain Identification of our Sample
* Here we identify the strain by using the following methods. We use basic dyes here to stain our
Sample to check whether the sample is Gram positive or not.
* We take a slide and drop a 100ul distilled water on it with the help of pipette and after that we
pick our culture with the help of loop and mix in it until it get evenly mix on it so for this we
mix it well with the help of loop.
* After mixing it evenly we heat fix it till it form a smear and we do this in laminar air flow.
* After that we pour Crystal violet on it for 1min and then wash it( so attain purple colour), and
then we pour Gram Iodine on it(so they retained the Stain) and then again wash it, then we pour
95% Ethanol on it(to decolorize it) and then again wash it, then we add Saffranine and wash it.
Then we leave it too air dry.
* What we see is after adding Ethanol some bacteria get decoloured and when we add Saffranine
on it all those bacteria acquired red colour which were decoloured.
* After that we check this on Microscope for strain Identification and through this process we are
able to Identify our bacteria.

12. Growth Kinetics of Bacteria
* Here we measure the growth of bacteria w.r.t time.
* So we add up Sucrose, NaCl, Yeast, MgSo4
, NaH2
Po4
, in 50ml of water in which also contain
NAM in Conical Flask after that we leave it in Autoclave for it to get sterilized.
* After that we inoculate it with 50ul of culture to the Conical Flask and from that we transfer 2ml
to two test tubes one is blank and the other is Not. We do all this process in Laminar Air Flow.
* Right after that we take the OD and put the flask back in shaker.
* And after every half hour we take the OD to check the different phases(Lag, Log extra).
* We stop until when we see no or decrease in the OD count. Either the count doesn’t increase or
started to decrease and by this we know that the death or decline phase start therefore we quit
taking any further OD.
* That’s how we are going to find the Growth Kinetics of our Bacteria.

13. Fermentation and DSP
• Now here we do fermentation and DSP to extract our Crude Enzyme. DSP stand for Downstream
processing to extract our Enzymes. But before extracting we have to purify our enzyme by Salt
and dialysis.
• So for this we have to make Nutrient agar in a flask and then we inoculated it with our Culture and
then we leave it for 1 night to get growth.
• After that we transferred it to 10 Appendrof tube 1.5ml in each tube and after that I centrifuged it
at 10,000 rpm for a period of 10minute.
• After centrifuging it I discarded the supernatant out then we add 1ml Tris buffer in the 1st
tube
and mix it well and then through pippete we take 1ml from the 1st
one and add it too 2nd
one
and I did the same in the rest of tubes.
• Then we collect all the Supernatant and add 3,2gm Ammonium Sulphate pinch by pinch in a
Magnetic stirrer. I both of them with the help of Magnetic bead and then we leave them for 10 min
and incubate it at 4℃ in a Dialysis bag.
• And 1 day of Incubation we do the Nattokinase estimation.

14. Lowry’s Standard Method for Protein Concentration
* Lowry method for protein concentration was done by mixing Folin Reagent in the protein sample
then leaving it for 30minutes and then taking the Optical Density of the sample with the help of
Spectrophotometer.
* While doing this we have to make certain reagents which are necessary for Protein concentration.
Like Reagent A== 2% Na2
Co3
+ 0.1 N NaOH
Reagent B== 0.5% CuSo4
+ 1% Potassium Sodium Tartrate
Reagent C == 49ml (A) + 1ml(B)
Reagent D == 3.5 ml(Dw) + _3.5ml (Folin’s Reagent)
* Now we are going to use all these Reagent for the Protein Estimation present in our Sample.
* We add all these Reagent in the test tube and after that we add distilled water in all the test tubes.
* And then we add Reagent C in all those tubes and leave it for 10 min and after that we add
Reagent D in all those test tubes then we leave it for 30 min in dark. After that we take the OD
with the help of Spectrophotometer.

15. S.NO Casein Dw Sample
1 0 1 0
2 0.1 0.9 0
3 0.2 0.8 0
4 0.3 0.7 0
5 0.4 0.6 0
6 0.5 0.5 0
7 0.6 0.4 0
8 0.7 0.3 0
9 0.8 0.2 0
10 0.9 0.1 0
11 1 0 0
12 0.5 0 0.5
TCA
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
RC
5
5
5
5
5
5
5
5
5
5
5
5
RD
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
OD 680
0.33
0.55
0.76
0.98
1.27
1.77
1.78
1.80
1.73
1.45
1.63
1.99
37℃
30mi
n
10min
30mi
n
dark
10min

16. Its an analytical method in which separation of active constituent in complex mixture.
This technique mainly done to purify, isolate and Identification and extraction of
compounds.
This method consist of two phases
1. Mobile phase
2. Stationery Phase
* So I separated the enzymes with the help of this technique, so that I can get a purified
form of Enzyme “Nattokinase”.
* HPLC have been used to both Quantify and Quality the product present on the
mixture.
* After that we get our Product from the sample and later we Identify it too by various
methods.
Purification by using HPLC

17. Figure: Shows Soil Sample
Figure: shows no. of Colonies after Spreading

18. Figure: shows Streaking in the Media

19. PM1 00.01
PM2 0.21
PM3 0.00
Figure: Shows the Concentration of Sample in the Test Tube

20. Dextrose 0.33
Maltose 0.42
Sucrose 0.56
Figure: Shows the Concentration of Sample in different Test tubes

21. Yeast 0.90
Peptone 0.31
Yeast +
Peptone
0.22
fBlank 0.16
pH4 0.02
pH7 0.77
pH9 0.39
pH11 0.35
Figure: Shows the Sample Growth at different PH concentration

22. Mgcl2 0.40
NaCl 0.50
CaCl2 0.07
ZnSo4
0.03
MgSo4
0.12
CuSo4
0.15
Figure: Shows the growth of Bacteria in different Components

23. K2
HPo4
0.47
Nah2
po4
0.65
KH2
po4
0.47
Figure: Shows the concentration of Sample when different Phosphate groups are added

24. Result:
So the strain is Gram Positive
Bacillus Subtilis.

25. OD 0 Min.
0.03
OD 30min
0.07
OD 60min
0.12
OD 90min
0.35
OD 120min
0.33
Growth Kinetics at different Time Interval

26. OD 150min
0.33
OD 180min
0.47
OD 210min
0.49
OD 240min
0.47
OD 270min
0.45

27. Figure: Shows the Protein Concentration in different test tubes in increasing Order

28. HPLC Results

29. THANK YOU