enzyme (2)

A
Training Report
On
“Advanced Biotech Techniques”
Submitted to:
Sam Higginbottom Institute of Agriculture,
Technology and Sciences
Submitted by:
Sapna Singh
For the partial fulfillment of the degree of
B. Sc. Biotechnology
Under guidance of
Dr. Vineeta Singh
(Sr. Research Scientist)
MRD LifeSciences
Lucknow

ACKNOWLEDGEMENT
I owe my soulful thanks to almighty for endowing his immense blessings that helped me
towards the successful completion of my training.
I express my deep sense of gratitude and indebtedness to George Thomas, H.O.D., Jacob
School of Biotechnology and Bioengineering, Sam Higginbottom Institute of Agriculture
Technology and Sciences, Allahabad, for his kind support.
I express my deep sense of gratitude and indebtedness to Mr. Manoj Verma, Director, and
Er. D. K. Verma, Project Manager, MRD LifeSciences, for his kind support and facilities
provided at this well famed Research Institute.
I wish to place on record my indebtedness to Dr Vineeta Singh ,Sr.Research Scientist, Mr
Abhinav Kumar Singh ,Research Scientist Ms. Sakshi Srivastava, Jr. Research Scientist
and Ms. Pallavi Sharma, Jr. Research Scientist, MRD LifeSciences, for their sustained co-
operation, interest and encouragement throughout this project/training work.
Sincerely I express my deep feelings to my friends who rendered a helping hand in the hour
of need.
I am indebted to my parents for their moral support and personal sacrifice to see me through
this project work.
Sapna Singh

DECLARATION
We hereby declare that the present work on “Enzymology” is a record of original work done
by us under guidance of Dr Vineeta Maam, Research Scientist, MRD LifeSciences, during
1st June 2016 to 30th June 2016, at MRD LifeSciences, Lucknow. All the data which were
provided in this were through our own work.
We also declare that no part of this thesis has previously been submitted to any University or
any examining body for acquiring any diploma or degree.
Date: 30/06/2016 Sapna Singh
Place:

INDEX
S.NO EXPERIMENT REMARKS
1 Bio-Instrumentationfor Wet Lab
2 Isolation of bacteria from starchy soil
sample
3 Purification of obtained mixed culture by
different streaking method
4 Screening of purified culture for amylase
production
5 Stain improvement by the two methods-
6 To study the Bacterial growth curve.
7 Enzyme Assay in crude enzyme by DNS
method.
8 Protein estimation by Bradford’s
method.
9 Protein estimation by Lowry’s method.
10 To perform fermentation.
11 To performs precipitation of enzyme by
salt precipitation method
12 To perform dialysis
13 To perform downstream processing of
submerged fermented culture.
14 To perform down streaming processing
of amylase solid state fermentation.
15 To perform SDS-PAGE

16 To study the effect of temperature, pH.
17 To Study the effect of Activator and
Inhibitor

EXPERIMENT NO-01
OBJECTIVE: - Isolation of bacteria from starchy soil sample.
REQUIREMENTS:- Test tubes ,soil sample,neck,distilled water,pipette,NA media and
petriplates,0.5gm soil, Normal saline (0.85% Nail ),Autoclave, inoculation needle
PRINCIPLE:-
Serial dilution:-The serial dilution agar plating method is one of the commonly used
procedures for the isolation and enumerates of bacteria. The method is bade upon the principle
when material containing bacteria is cultured, each viable organism will develop in colony ,and
serial dilution is done in order to reduce the number of bacteria enumerate is done in order to
increase in the population.
Pouring: - Agar media was poured in half of petriplate, agar was solidifying agent which helps
in microbial growth.
Spreading: - The spread plate technique is used for the separation of dilution, mixed
population of bacteria so that individual colony can be obtained. In this technique microbes
are spread over the solidified agar medium with a sterile L shape rod.
PROCEDURES:-
 0.5 gm of soil sample was suspended in 50 ml of normal saline and mixed thus
stock solution formed.
 From the stock solution 0.5 gm of normal was pipette out and poured in the
second test tube containing 4.5 ml of normal saline. Now the second test tube
mixed.
 Each sample was serially up to 5 test tube and the dilution rate are 10-1,10-2,10-3,
10-4 and 10-5respectively. From last test tube, we discard 0.5ml to maintain the
volume.
 From last three dilutions, 50 µl solutions was pipette out and spreads on the surface
of sterile petriplate of different prepared solidified agar media by spread plate’s
method.
 Now petriplate were kept for incubation for 24 hours at 37⁰C.
OBSERVATION: - After proper incubation period, number of colonies on the plates.

Figure: 1:-Serial dilution (a) 10-3 (b) 10-4 (c) 10-5
RESULT: Different bacterial colonies were seen in the NAM plates. Bacteria from soil were
isolated by serial dilution method.
Table:-Physical and morphological characteristics of the isolated colonies (10-5)
COLONY
MORPHOLOGY
COLONY NO-01 COLONY NO-02 COLONY NO-03
SHAPE Circular Circular Rhizoid
MARGIN Discrete Curled Curled
ELEVATION Flat Flat Convex
PIGMENTATION Off White Off White Off White
SURFACE Smooth Smooth Smooth
TEXTURE Soft Soft Soft
OPACITRY Opaque Opaque Opaque
COLOUR Off White Off White Off White
10-5
10-4 10-3

EXPERIMENT NO-02
OBJECTIVE: - Purification of obtained mixed culture by different streaking method.
REQUIREMENTS: - Culture, Petriplate, Spirit lamp and inoculating loop.
PRINCIPLE: -Pure Culture is a culture containing colony of single bacteria. Pure culture is
obtained by streaking and quadrant streaking is the best method of streaking to obtain pure
culture
TYPES OF STREAKING:-
 Zig Zag
 Continuous Quadrant
 Discontinuous Quadrant
PROCEDURE:-
 NAM (Nutrient agar media) was prepared and autoclaved.
 Media was poured and allowed to solidify.
 After solidification, isolates colony to 01, 02, and 03 respectively (from 10-5) by
an inoculating loop and streaked on solidified nutrient agar plates.
 Incubate the inoculated plate at 37⁰C for 24 hours.
OBSERVATION: - As the Striking precedes cell number decreases.
Figure: - Pure Culture Plates
RESULT: - Pure Culture is obtained.

EXPERIMENT NO:-03
OBJECTIVE: - Screening of purified culture for amylase production.
REQUIREMENTS: - Minimal agar medium, pure culture, starch, Petriplate, spirit lamp,
inoculating loop.
PRINCIPLE: - Screening technology involves deletion and isolation of high yielding species
from sources such as soil containing amylase producing bacteria. Through screening potential
microbes were isolated. Screening for amylase production is performed in minimal agar media
which has very limited amount of carbon source( glucose is lacking 0.5% )MAM for amylase
is supplemented with 1% starch which is only available source of carbon for bacteria after it
has consumed the limited amount of glucose present in the media .Now only those bacterial
species will be able to grow which has the capability of producing amylase which can
hydrolyses starch present in the media, amylase is further screened by the help of starch ,
where core zone of hydrolysis is seen.Screening technique can be divided into two levels
 Primary Screening
 Secondary Screening
Primary Screening: - is used to check the growth of bacteria in minimal agar media
under stress conditioned by visual identification.
Secondary Screening: - add iodine solution.
Prepare 100ml MAM + 1% Starch
COMPOSITION: -Composition of Minimal Agar media (MAM)
 Potassium Dehydrogenate phosphate 3.0g
 Disodium hydrogen phosphate 6.0g
 Sodium Chloride 5.0g
 Ammonium Chloride 2.0g
 Magnesium Sulphate 0.1 g
 Glucose/Dextrose 8.0g
 Supplemented with Starch 10.0g
 Distilled water 1000ml
 pH 7
 Agar 15g

PROCEDURE:-
 MAM was prepared and autoclaved.
 Pour the media in sterile plates and allow it to solidify.
 Central Striking was performed with 01, 02, 03 colonies culture on solidified
MAM plates.
 Incubate the plates for 2 days at 37⁰C.
 After incubation the plates were flooded with iodine.
 Plate was observes for core zone formation.
OBSERVATION: -Core Zone formation is seen.
OBSERVATION TABLE:-
S.NO COLONY RESULT
1. 01 ++
2. 03 ++
3. 09 +++
Figure:-Amylase Screening
RESULTS: - The cultures show positive Screening.

EXPERIMENT NO-04
OBJECTIVE: - Stain improvement by the two methods-
 Physical mutation-Stain improvement by UV
 Chemical mutation-Stain improvement by EtBr
Physical Mutation
REQUIREMENT: - Bacterial Culture with NB Media, NA Media (pH 7), Petri plates, LAF,
Spreader.
PROCEDURES:-
 100ml NAM was prepared, autoclaved, pour and allow solidifying.
 3 Plates of NAM were prepared.
 Plates mentioned as control, 1mins, and 3mins, 9mins, and UV treatment.
 20µl of the inoculums was spread on respective plates.
 We gave UV treatment respectively to all NAM Petri plates.
 Then incubated all plates at 37⁰C for 24 hrs.
 After incubation the UV treatment was done.
 Performed ENZYME ASSAY.
RESULT: - The isolated bacterial sample was subjected to different UV treatment condition
to identify the most suitable condition for the growth of the culture. The treatment is serially
given as control, 1mins, 3 mins, and 9minsrespectively. This table has shown the duration
and isolated colony of the culture after UV treatment.
S.NO DURATION OF UV COLONY COUNT
1. Control Lawn
2. 1mins Lawn
3. 3mins Lawn
4. 9mins Lawn

Figure:-U.V treated bacteria
Chemical Mutation
REQUIREMENT: - NB media, EtBr, Test tube, Pipette, UV treated bacterial Culture
PROCEDURE:-
 Prepare 20 ml of NB.
 Transfer 3 ml of NB media in each tube.
 Test tubes are mentioned as control 1µl/ml, 2µl/ml, 3µl/ml, 4µl/ml, 5µl/ml
respectively.
 Autoclave all Tests tubes and cool.
 Add calculated amount of EtBr in each Test tube and discard same volume of
NB and mix well.
 Then inoculate 20µl of bacteria in each test tube.
 Incubate 24 hrs in incubator.
 After the incubation the bacterial culture was spread on the NAM respectively,
name as control, 1µl/ml ...............5µl/ml.
 Incubate 24 hrs in the incubator.
 Colony of bacteria was absorbed in the plate to show the good bacterial culture.
RESULT: -The isolated bacteria simple was subjected to different EtBr treatment condition to
identify the most suitable condition for the growth of the culture. The treatment is serially
given as control, 1µg/ml.................. Respectively. This table showed the concentrating of EtBr
and isolated colony of the culture after EtBr treatment.
S.NO CONC.OF EtBr COLONY COUNT
1. Control Lawn
2. 1µl/ml Lawn
3. 2µl/ml Lawn
4. 3µl/ml Lawn

5. 4µl/ml Lawn
6. 5µl/ml Lawn
Figure:-EtBr treated Plate

EXPERIMENT NO: 05
OBJECTIVE: To study the Bacterial growth curve.
Principle: Growth curve may be defined as orderly increase of all the components of living
cells, its size and results in multiplication of cells. Bacterial growth is studied by analyzing
the growth curve of a microbial culture. When micro-organisms are grown in a closed system
or batch culture, the resulting growth curve usually has 4 phases;
1. Lag phase: When the cells are adjusting their new environment. During this phase,
cellular metabolism is accelerated, resulting in rapid biosynthesis of cellular
macromolecules, primarily enzymes in preparation for the next phase of the cycle.
Although the cells are increasing in size. There is no cell division and increase in no.
2. Log phase: Under optimum nutritional and physical conditions, the physiologically
robust cells reproduce at a uniform and rapid rate by binary fission. Thus there is a
rapid exponential increase in population, which doubles regularly until a maximum
no. of cells are reached. The length of the log phase varies, depending upon the
organism and the composition of medium, although the average may be estimated to
last 6 to 12 hrs.
3. Stationary phase: during this stage no. of cells undergoing division is equal to the
no. of cells that are dying. There is no further increase in cell no. and the population is
maintained at its maximum level for a period of time.
4. Decline phase: Because of the continuing depletion of nutrients and build up of
metabolic wastes, the microbes die at a rapid and uniform rate. This decrease in
population closely parallels its increase during the log phase. However small no. of
highly resistant organism persists for an intermediate length of time.
Requirements: NB, broth culture, inoculation loop, pipette
Procedure:
 100ml NB was prepared in a flask.
 Autoclaved and cools it.
 Inoculate with 100µl of broth (broth was prepared from 2min U.V treated P.P.).
 Incubate @ 37Ċ/120rpm for 7 days in shaker.
 Read O.D @ 600nm after every 24 hr up to 7 days.
 For O.D reading we require blank to set the zero. So we prepared 5ml NB, heat for
5sec, cool it and filled in cuvett.

OBSERVATION:-
Day O.D at 540 nm
1 0.02
2 0.08
3 0.12
4 0.13
5 0.13
6 0.12
7 0.10
Result: Growth curve was obtained.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Day 1 Day2 Day 3 Day 4 Day 5 Day 6 Day 7
O.Dat540nm
Time

EXPERIMENT NO-06
OBJECTIVE: - Enzyme Assay in crude enzyme by DNS method.
REQUIREMENTS: -Test tube, fermented broth, 1% Starch, DNS Reagent, Distilled water
and pipette.
For DNS Reagent:-
 DNS
 Sodium Potassium Tartarate
 2N NaOH
DNS STANDARD GRAPHS:-
Standard graph was plotted to calculate the enzyme activity by the help of standard graph we
can calculated the amount of maltose released by comparing the O.D. This table shows the
different concentration of starch. By helping of these reading we plotted the standard graph
shown in figure.
Figure: - DNS Standard

Table: DNS Standard Graph
S.NO Maltose
0.5 mg/ml
Distilled
water
Conc. of
maltose in
mg/ml
DNS
reagent
in (ml)
Water
Bath
15 min
in
100⁰C
Distilled
water
V
O
R
T
E
X
O.D
at 540
nm
1 0.0 1.0 0.0 1 5 0.00
2 0.1 0.9 0.05 1 5 0.03
3 0.2 0.8 0.10 1 5 0.07
4 0.3 0.7 0.15 1 5 0.13
5 0.4 0.6 0.20 1 5 0.19
6 05 0.5 0.25 1 5 0.24
7 0.6 0.4 0.30 1 5 0.28
8 0.7 0.3 0.35 1 5 0.34
9 0.8 0.2 0.4 1 5 0.38
10 0.9 0.1 0.45 1 5 0.42
11 1.0 0.0 0.50 1 5 0.46
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5
O.Dat540nm
Conc of maltose (mg/ml)

DNS ASSAY FOR U.V TREATED BACTERIA:-
S.N0 Time (min) O.D at 540 nm
1 6 0.10
2 9 0.13
3 15 0.20
DNS ASSAY FOR EtBr TREATED BACTERIA:-
S.NO Test Tube O.D at 540 nm
1 Control 0.21
2 1µl 0.12
3 2µl 0.11
4 3µl 0.09
5 4µl 0.08
6 5µl 0.04
0
0.05
0.1
0.15
0.2
0.25
6 9 15
O.Dat540nm
Time of Exposure

0
0.05
0.1
0.15
0.2
0.25
control 1µl 2µl 3µl 4µl 5µl
O.Dat540nm
Concentration of EtBr

EXPERIMENT NO:-07
OBJECTIVE: - Protein estimation by Bradford’s method.
REQUIREMENT: - BSA, Bradford’s reagent, Distilled Water, 11 Test Tube, Pipettes and
Flask.
Composition of Bradford’s reagent:-
 Coomasie Brilliant Blue G-250 (CBB G-250)
 Orthophosphoric acid 85%
 Ethanol 95%
PRINCIPLE:-
Bradford’s assay is very fast. The assay is based on the observation that the absorbance
maximum for an acid solution of Comassie Brilliant Blue G-250 shift from 465 nm to 595 nm
when binding to protein occurs .Both Hydrophobic anionic interaction stabilizes the anionic
form of dye, causing a visible colour change. Under acidic condition form of dye is converted
into its blue form. The binding of dye to protein stabilizes the blue anionic form. Increase in
absorbance at 595 nm is proportional to amount of bound dye which is proportional to
amount of protein present in the sample.
PROCEDURE:-
Preparation of Bradford’s reagent:-
 0.003 gm of Coomasie Brilliant Blue G-250 was added in 5ml of 95% ethanol.
 After that 5 ml of 85% orthophosphoric acid was added. The mixture is stirred
for approximately 10 mins. Then solution is diluted to 50 ml distilled water and
filtered.
Bradford micro assay for Total Proteins;-
 Take 10 test tubes along with the blank in increasing order of BSA.
 In 1sttest tube 0.0 and 500µl, make the final volume 500µl with the help of
distilled water.
 Add 2.5 ml of Bradford reagent in each test tube, vortex and keep the test tubes
for 10 mins at room temperature.

Take the O.D. at 595nm and the plot the graph between protein concentration
(BSA) vs Absorbance.
OBSEVATION TABLE-
RESULT: - Concentration of unknown sample is determined by using standard.
Test
Tube
Volume of
Standard
BSA (ml)
Volume
of
Distilled
water
(ml)
Conc. Of
BSA
Volume of
Bradford
regent (ml)
Incubate
in
Dark
for
10 min.
V
O
T
E
X
O.D.at
595nm
1 0.0 0.50 0.00 2.5 0.00
2 0.05 0.45 0.02 2.5 0.21
3 0.10 0.40 0.04 2.5 0.36
4 0.15 0.35 0.06 2.5 0.51
5 0.20 0.30 0.08 2.5 0.56
6 0.25 0.25 0.10 2.5 0.63
7 0.30 0.20 0.12 2.5 0.72
8 0.35 0.15 0.14 2.5 0.79
9 0.40 0.10 0.16 2.5 0.86
10 0.45 0.05 0.18 2.5 0.93
11 0.50 0.0 0.20 2.5 0.98

EXPERIMENT NO:-08
OBJECTIVE- Protein estimation by Lowry’s method.
REQUIREMENTS- BSA, reagent A, reagent B, reagent C, reagent D, distilled water, flask
and pipette.
REAGENT A- 2% Na2CO3 in 0.1 N NaOH
REAGENT B- 0.5% CuSO4 in 1% sodium potassium tartarate
REAGENT C- 50 ml of REAGENT A + 1ml of REAGENT B
REAGENT D- F.C and D.W. [1:1]
STOCK BSA – 0.5 mg/ml
Working BSA- 1:4
PRINCIPLE-
The phenolic group of tyrosine and tryptophan residues in a protein will produce blue purple
colour complex i.e. heteropolymolybdate and heteropolytungstate with maximum absorption
in the region of 660 nm wavelength with Folin-Ciocalteau reagent which consists of
phosphomolybdic acid and phosphotungstic acid, thus the intensity of colour depends on the
amount of theses aromatic amino acids present and will thus vary for different proteins. Most
proteins estimation techniques use Bovine Serum Albumin (BSA) universally as a standard
protein because of its low cost, high purity and ready availability. This method is sensitive to
down about 10µg/ml and is probably the most widely used protein assay despite it being only
a relative method, subject to interference from Tris buffer, EDTA, non-ionic and cationic
detergents, carbohydrate, lipids and some salts. The incubation time is very critical for a
reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5.
PROCEDURE-
 11 testubes were taken along with blank in increasing order of the BSA from 0.1 to
1.0 ml
 Final volume was made up to 1ml with the help of distilled water.
 Now 5 ml reagent C is added in each of the tube, vortexed and kept aside for 10 mins.
 After 10 mins 0.5 ml of reagent D is added in each of the testube and kept in dark
chamber for half an hour at room temperature.

 Zero the colorimeter with the blank and took OD at 660nm.
 Plotted the absorbance against protein concentration to get a standard calibration
curve.
 Checked the absorbance of unknown sample and determine the concentration of the
unknown sample using the standard curve plotted
OBSERVATION-
BSA
WORKING
(ml)
DISTILLED
WATER
(ml)
CONC.
OF
BSA
(mg/ml)
REAGENT
C
(ml)
REAGENT
D
(ml)
O.D.
AT
660
nm
0.0 1.0 0.00 5 INCUBATE 0.5 INCUBATE V 0.0
0.1 0.9 0.02 5 AT 0.5 IN 0.02
0.2 0.8 0.04 5 ROOM 0.5 DARK O 0.07
0.3 0.7 0.06 5 TEMP. 0.5 CHAMBER 0.10
0.4 0.6 0.08 5 FOR 0.5 FOR R 0.16
0.5 0.5 0.10 5 10 0.5 30 0.19
0.6 0.4 0.12 5 MINS 0.5 MINS T 0.23
0.7 0.3 0.14 5 0.5 0.27
0.8 0.2 0.16 5 0.5 E 0.28
0.9 0.1 0.18 5 0.5 0.31
1.0 0.0 0.20 5 0.5 X 0.36

RESULT – Absorbance of the unknown sample and the concentration of the known sample
are determined using the standard curve plotted above.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
O.DAT660NM
CONCENTRATION OF BSA

EXPERIMENT NO. - 09
OBJECTIVE – To perform fermentation.
PRINCIPLE- Fermentation is production of industrially important products by the help
of microorganism under controlled physical and chemical condition.
Figure: - Fermentor
 PH sensor- it is to maintain the pH.
 Temperature probe – to maintain temperature.
 Filter- its pore size is 0.2-0.5µ.
 Impeller cum sparer- it contain fan like structure which is used to mix the content
and it contain some pores through which air can circulate in whole fermentation.
 Baffle – it’s to mix the content present in between the fermentor which is not
properly mixed by sparer.
 Sampling –it’s to take out the content.
Fermentation is of two types-
1. Submerged state fermentation
2. Solid state fermentation

1. Submerged state fermentation
Composition of production medium
NH4CL - 10gml
KH2PO4 - 1gml
MGSO4 - 0.04gml
NaCl - 5gml
Starch - 1.5%
Distilled water - 1000ml
PROCEDURE
 100ml of production media was prepared and autoclave.
 Cultures are inoculated in the prepared broth.
 Flask is kept in orbital shaker for 4 days at 37°C.
Solid state fermentation
Composition of MSM (minimal salt media)
NaH2PO4 - 3gml
Na2HPO4 - 6gml
NaCl - 5gml
NH4Cl - 2gml
MgSO4 - 0.1gml
Glucose - 8gml
pH - 7
Distilled water - 10
PROCEDURE:-
 10 gm sugarcane waste was taken in conical flask
and moistens it with 25 ml MSM.

 Autoclave and cool it.
 After the 1 ml of 4 minute culture is inoculated in the flask with the help of pipette.
 Now flask is kept in incubator at 37°C for 4 days.
OBSERVQATION – Microorganism was growing in flask by using nutrient and carry out
fermentation.
RESULT:-Fermented product was obtained.

EXPERIMENT NO. – 10
OBJECTIVE- To performs precipitation of enzyme by salt precipitation method.
PRINCIPLE- This is the most common way to precipitate protein. Precipitation is done by
two method- salt and solvent precipitation. Salt precipitation is better, because in solvent
precipitation denaturation of protein takes place.
PROCEDURE:-
 Take a beaker containing 21.5ml of submerged crude enzyme and 10ml of solid state
crude enzyme & surrounded it in a container maintained ice at 4°C.
 Plays the whole system on the magnetic stirrer.
 Take 10.15gm ammonium sulphate for submerged crude enzyme and 4.762gm for
solid state crude enzyme & Add pinch by pinch after every 4-5 minutes.
 Store over night.
 Transfer the sample solution in micro-centrifuge tube and spin at 10000 rpm 10min
for 4°C. Discard supernatant and store pellet.
 Take the pellet and add 1 ml Tris HCL to dissolve the pellet and make the final
volume 20ml of 100mM tris HCL.
 Then perform dialysis.
RESULT: -Enzyme has been precipitated.

EXPERIMENT NO-11
OBJECTIVE- To perform dialysis.
PRINCIPLE-
The concentrated protein solution obtained after salt precipitation is placed in the dialysis bag
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.
Figure: -Submerged fermentation
REQUIREMENT MATERIAL:
10ml (submerge protein), dialysis bag, thin thread, D/W, 1%SDS, 100mMtris HCL
PRETREATMENT OF BAG:
PROCEDURE:
 Dialysis bag boil for 10 minute in D/W (1×2), and boil for 10 minutes in .1% SDS
(1×2), then boil for 10 minutes in D/W (1×2).
 Dialysis bag dry for tissue paper and fill the protein in them then knot for thin bread.
 Dialysis bag place 200ml 100mM tris HCL pH 8 in beaker and buffer change 2 to 3
time more, store at 4˚c for overnight.
OBSERVATION: Due to endo-osmosis bag become tough.
RESULT: Dialysis test has been performed successfully

EXPERIMENT-12
OBJECTIVE- To perform downstream processing of submerged fermented culture.
REQUREMENTS-Fermented broth, funnel, flask, eppendorf tube, measuring cylinder and
pipette.
PRINCIPLE-Downstream processing refers to the recovery and purification of biosynthesis
products, particularly pharmaceuticals from natural sources such as animal or plant tissue or
fermented broth, including the recycling of salvageable components and the proper treatment
and disposal of wastes. It is an essential step in manufacturing of pharmaceuticals such as
antibiotics, hormones, antibodies, and vaccines, antibiotics and enzymes used in diagnostic
industrial enzymes and natural fragrance and flavor compounds. It is usually considered a
specialized field in biochemical engineering itself a specialization within chemical
engineering though many of key technologies were developed by chemists and biologists for
laboratory scale report of biological products.
PROCEDURE-
 Take desired volume from PM containing NH4Cl-10gm/liter(by retaining peptone+
yeast extract)-10gm/liter, starch1.5%,MgSO4-0.04%(by replacing Ca and Mg
respectively)
 Named that media as OPM (optimized production media)and prepare media for
100ml.
 Autoclave then cool.
 Incubate 1ml of bacteria.
 The flask was incubated in shaker at 150 rpm at room temperature for 4 days.
 Submerged fermented broth was taken from orbital shaker at and centrifuges it at
5000 rpm for 5mins at 40C.
 Supernatant was collected and treated as crude enzyme.
 This crude enzyme is retained at 40C for further use.
 Pellets are kept at 40C for extraction of intracellular amylase.
OBSERVATION- Crude enzyme is obtained and its volume is measured used for
precipitation and determining total proteins, total activity and specific activity.

PROTEIN ESTIMATION BY LOWARY AFTER DSP-
Test
tube
Enzyme(ml
)
D/W(ml
)
Reagen
t C(ml)
Reagen
t D(ml)
V
O
OD at
640n
m
Blan
k
0.00 1.00 5 Incubate at 0.5 Incubat
e at
dark
R
T
0.00
Test 0.50 0.50 5 Room
temperatur
e
0.5 For 30
min
E
X
1.20
ENZYME ACTIVITY BY DNS AFTER DSP-
Test
tube
Starch(m
l)
Enzyme(m
l)
D/W(m
l)
Incubat
e for
DNS(m
l)
Incubat
e at
D/W(m
l)
V
O
OD at
540n
m
Blan
k
0.0 1.0 1 15min 1 1000C 5 R
T
0.00
Test 0.5 0.5 - at 370C 1 For 15
min
5 E
X
0.36

EXPERIMENT NO-13
OBJECTIVE: To perform down streaming processing of amylase solid state fermentation.
PRINCIPLE:
Solid state fermentation has been defined as the fermentation process occurring in the
absence of free water .Solid state fermentation processes generally employ a natural raw
material as carbon and energy source. Wheat bran which is most commonly used substrate in
solid state fermentation is obtained in two forms, fine and coarse.
REQUIREMET: - MSM (Minimal salt media) 25ml, conical flask.
PROCEDURE:
 Prepare 100ml MSM (Minimal salt media) and moist 10gm wheat bran and then
autoclave.
 Then inoculate 1ml of 4min.UV bacteria treated 3 days.
 Wheat bran 10gm+50ml 100mM tris HCL buffer, shake 120rpm for 1-2hr.
 Centrifuge at 5000/5 min, for 4˚c, collect supernatant extract as crude extract, use for
protein estimation and DNS assay.
OBSERVATION TABLE: Crude amylase is obtained and is measured used for
precipitation and determining total protein, total activity and specific activity.
RESULT: Total volume of crude enzyme is 10ml.

Protein estimation by Lowry’s method after DSP
Test
tubes
Enzyme(ml) D/W(ml) Reagent
C(ml)
Incubate
at room
temp for
10 min
Reagent
D(ml)
Incubate
at dark
for 30
min
V
O
R
T
E
X
OD at
640nm
Blank 0.00 1 5 0.5 0.00
Test 0.5 0.50 5 0.5 1.31
Enzyme activity by DNS after DSP
Test
tubes
Starch(
ml)
Enzyme(
ml)
D/W(m
l)
Incuba
te at
37˚ 15
min
DNS(m
l)
Wate
r
bath
15
min
D/W(m
l)
V
O
R
T
E
X
OD AT
540nm
Blank 0.00 0.00 1 1 5 0.00
Test 0.5 0.05 1 5 0.52

EXPERIMENT-14
OBJECTIVE- To perform SDS-PAGE
PRINCIPLE- SDS is an anionic detergent which binds strongly and denatures the protein.
The number of SDS molecules binding to a polypeptide chain is approximately half the
number of amino acid residues in the chain. The protein SDS complex carries net negative
charge, hence move towards the anode and separation is based on size of protein.
REQUIREMENTS- Stacking gel, separating gel, 10% SDS, 30% acrylamide mixture,
TEMED, 1M Tris HCL, 1% ammonium per sulphate, distilled water.
COMPOSITION – A. GEL
COMPOSITION SEPERATING GEL
(10%)
STACKING GEL
(4%)
30% Acrylamide 3.1ml 0.56ml
1M Tris HCL 2.5ml 0.65ml
D/W 3.3ml 3.5ml
10% SDS 100µl 50µl
10% APS 100µl 50µl
TEMED 15-30µl 10-15µl
B. SAMPLE
Take 1:1 volume 50µl sample and 50µl buffer. Incubate at 60⁰C for 5 mins, cool and load.
SAMPLE BUFFER-
Bromophenol blue
SDS
B- mercaptoethanol
Glycerol/Sucrose
C. LOADING AND ELECTROPHORESIS-
Movement in presence of electric supply

Electric buffer-
10% SDS-PAGE buffer
1.96mM glycine
500mM Tris HCL
1% SDS
D. STAINING SOLUTION-
0.2% C.B.B.R-250
Methanol
Glacial acetic acid
D/W
E. Detaining solution: for 100ml
20ml methanol
5ml glacial acetic acid
25ml D/W
PROCEDURE-
 Prepare stacking and separating gel.
 Continuous system- only separating gel 100% is filled in the plate.
 Discontinuous system- separating gel 70% and stacking gel 30 % is filled in another
plate.
 Place the comb above the running plate and base containing gel.
 Prepare sample buffer and electrode buffer.
 Take 3 eppendorf tubes.
 Take 50µl volume of sample BSA mg/ml and 50µl of LSB sample in 1st eppendorf.
 Take 50µl volume of sample LSB and crude enzyme (solid state fermentation) after
dialysis in 2nd eppendorf.
 Remove the comb from the plate.
 Fill the running tank with the electric buffer.

 Take 20µl from the 1st eppendorf tube and pour it in 1st two wells.
 Take 20µl from the 2nd eppendorf tube and pour it in rest of the wells.
 Connect the electrode system at 70v and place it in the fridge for 30 minutes.
 After 30 mins take it out from the fridge and keep it at R.T. at 5v for 15 mins.
 Take out the gel and keep it in staining solution for overnight.
 After 24 hrs wash the gel with the destaining dye.
OBSERVATION-

EXPERIMENT NO-15
OBJECTIVE- To study the effect of temperature, pH.
REQUIREMENTS- DNS reagents, test tube, NaOH, distilled water.
PROCEDURE-
1) For temperature-
 Take 5 test tubes and label as blank, T1 (22⁰C), T2 (37⁰C), T3 (R.T), T4
(50⁰C).
 Add 1 ml distilled water in 1st test tube.
 Take 0.5 ml enzyme in 2nd test tube and 0.5 ml 1 % starch and incubate at
20⁰C for 15 mins. Add 1 ml DNS reagent and boil, then add 5 ml D/W, take
O.D.
 Take 0.5 ml enzyme in 3rd test tube and add 0.5 ml 1% starch and incubate at
37⁰C for 15 mins. Add 1ml DNS reagent and boil, and then add 5 ml D/W.
Take O.D.
 Take 0.5 ml enzyme in 4th test tube and add 0.5ml 1% starch and incubate at
50⁰C for 15 mins Add 1ml DNS reagent and boil, then add 5 ml D/W. Take
O.D.
 Take 0.5 ml enzyme in 5th test tube and add 0.5ml 1% starch and incubate at
R. T.For 15 mins Add 1ml DNS reagent and boil, then add 5 ml D/W. Take
O.D.
Figure:-Effect of temperature

Observation table(for temperature):-
Temperature in ⁰C O.D at 540 nm
Submerged
fermentation
Enzyme
activity
Solid state
fermentation
Enzyme
activity
Blank 0.00 0.00 00 .00
22⁰ 0.23 0.05 0.03 .03
37⁰ 0.27 0.06 0.03 .032
Room temperature 0.21 0.04 0.03 .032
50⁰ 0.13 0.03 0.03 .04
2) For pH
 Take 5 test tube labels as blank,1,2,3,4.
 Set blank as 1st test tube.
 Take 0.5 ml enzyme in 2nd test tube and 0.5 ml 1% starch and maintain pH (5)
incubate at 37⁰C for 15 min. Add 1 ml DNS reagent and boil then add 5 ml D/W
.Take O.D.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
22 37 R.T 50
EnzymeActivity
O.D at different temperature
Submerged
Solid State

 Take 0.5 ml enzyme in 3rd test tube and 0.5 ml 1% starch and maintain pH (7)
.Take O.D.
 Take 0.5 ml enzyme in 4th test tube and 0.5 ml 1% starch and maintain pH (9)
.Take O.D.
 Take 0.5 ml enzyme in 5th test tube and 0.5 ml 1% starch and maintain pH (11)
.Take O.D.
OBSERVATION TABLE:-
pH O.D. at 540nm
Submerged
fermentation
Enzyme
activity
Solid state
fermentation
Enzyme
activity
0.00 0.00 0.00 0.00
5 0.16 0.037 0.08 0.01
7 0.31 0.07 0.29 0.06
9 0.26 0.06 0.17 0.02
11 0.55 0.12 0.44 0.09

RESULT: -The optimum temperature for activity of amylase is 11⁰C.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
5 7 9 11
EmzymeAssay
O.D at different pH
Submerged
Solid State

EXPERIMENT NO:-16
OBJECTIVE:-To study the effect of Activator and Inhibitor on amylase.
REQUIREMENTS-Mg++, Ca++, SDS, EDTA, Testtube, distilled water.
PROCEDURES-
1) For Activator:-
 Take 4 test tubes.
 Set 1st test tubes as blank and add D/W.
 Set 2nd test tube without activator.
 Take 0.5ml enzyme in 3rd test tube and 0.5 ml 1% starch and 0.2 ml Ca++
incubate at 37⁰C for 15mins .Add 1ml DNS and boil then add 5 ml D/W. Take
O.D.
 Take 0.5ml enzyme in 4th test tube and 0.5 ml 1% starch and 0.2 ml Mg++
O.D.
OBSERVATION TABLE (activator) –
pH O.D. at 540nm
Submerged
fermentation
Enzyme
activity
Solid state
fermentation
Enzyme
activity
Blank 0.00 0.00 0.00 0.00

Without Activator 0.14 0.03 0.13 0.03
Ca++ 0.13 0.03 0.13 0.03
Mg++ 0.21 0.04 0.22 0.05
2) For Inhibitor:-
 Take 4 test tubes.
 Set 1st test tubes as blank and add D/W.
 Set 2nd test tube without activator.
 Take 0.5ml enzyme in 3rd test tube and 0.5 ml 1% starch and 0.2 ml SDS
incubate at 37⁰C for 15mins .Add 1ml DNS and boil then add 5 ml D/W.
Take O.D.
 Take 0.5ml enzyme in 4th test tube and 0.5 ml 1% starch and 0.2 ml EDTA
O.D.
0
0.01
0.02
0.03
0.04
0.05
0.06
Without Activtor Mg Ca
Enzymeactivity
O.D with different activator
Submerged
Solid State

OBSERVATION TABLE (Inhibitor):-
Inhibitor O.D. at 540nm
Submerged
fermentation
Enzyme
activity
Solid state
fermentation
Enzyme
activity
Blank 0.00 0.00 0.00 0.00
Without Inhibitor 0.21 0.04 0.13 0.03
SDS 0.20 0.04 0.12 0.03
EDTA 0.12 0.03 0.13 0.03
RESULT:-The enzyme activity is enhanced by Mg2+ and deactivated by SDS.
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0.045
Without
Inhibitor
EDTA SDS
EmzymeAssay
O.D with different Inhibitor
Submerged
Solid State

enzyme (2)

More Related Content

What's hot (20)

Similar to enzyme (2) (20)

enzyme (2)