Advanced Bioprocess and Downstream-III Sem PG

KARPAGA VINAYAGA COLLEGE OF ENGINEERING AND TECHNOLOGY

Chinnakolambakkam - 603308

DEPARTMENT OF BIOTECHNOLOGY

BY7312-ADVANCED BIOPROCESS AND DOWNSTREAM PROCESSING LAB

Prepared by.Mrs M.Srividhya

Assistant ProfessorDepartment of Biotechnology,

Karpaga Vinayaga College of Engineering and TechnologyChinnakolambakkam-603308,

BY7312-ADVANCED BIOPROCESS AND DOWNSTREAM PROCESSING LAB

LIST OF EXPERIEMENTS

S.NO EXPERIEMENT PAGE NO.

1. BATCH CULTIVATION 02

2. THERMAL DEATH KINETICS 04

3. CELL AND ENZYME IMMOBILIZATION 06

4. ESTIMATION OF KLa BY SULPHITE

OXIDATION METHOD

08

5. RESIDENCE TIME DISTRIBUTION 10

6. DETERMINATION OF OVERALL HEAT

TRANSFER COEFFICIENT

12

7. ESTIMATION OF KLA BY DYNAMIC GASSING

OUT METHOD

14

8. FED BATCH REACTOR 16

9. ESTIMATION OF KLa BY POWER CORRELATION METHOD

18

10. DETERMINATION OF POWER REQUIREMENT FOR BATCH REACTOR

20

11. EFFECT OF TEMPERATURE ON AMYLASE ACTIVITY

22

12. EFFECT OF pH ON AMYLASE ACTIVITY 26

13. DETERMINATION OF ENZYME KINETICS PARAMTERS BY VARYING SUBSTRATE CONCENTRATIONS

29

14. ION EXCHANGE CHROMATOGRAPHY 33

15. CENTRIFUGATION 36

16. ALKALINE LYSIS 38

17. ULTRASONICATION 40

Experiment No: 1

Date :

BATCH CULTIVATIONAIM

To run the batch reactor for the growth of yeast and hence to calculate the growth rate

and doubling time.

PRINCIPLE

Batch process operates in close system. Substrate is added at the beginning of the

process and products removed only at the end of the fermentation. Most commercial

bioreactors are mixed vessels operated in batch. The classic mixed reactors is the stirred tank

reactor (CSTR) .However, mixed reactors can also be bubble column, air lift reactor or other

types. The cost of running a batch reactor depends on the time taken to achieve the desired

product concentration for a batch reactor from distinct phases of growth are present; lag phase,

exponential phase, stationary phase and death phase.

The lag phase occurs immediately after inoculation and is a period of adaptation of

cells to a new environment. Depending on the concentration of nutrients, new enzymes are

synthesized and the synthesis of other enzymes are suppressed and the internal machinery of

the cell is adapted to the new environmental conditions.

Following the lag phase period, growth starts in the acceleration period and continues

through the growth and decline phase. If growth is exponential it appears as a straight line on a

semi log graph. As a nutrient in a culture medium becomes depleted or inhibitory, product

accumulates, growth slows down and the cell enters the decline phase. After the transition

period, the stationary phase is reached during which no further growth occurs. Some cultures

exhibit second exponential phase after the first stationary phase and the growth is called dioxic

phase of growth.

PROCEDURE

1. Two liters of potato dextrose medium was prepared.

2. The pH and DO probes were calibrated.

3. The medium was transferred into a reactor and sterilized at 121°C for 20 minutes.

4. The medium was cooled and brought to 37°C.

5. The pH, temperature, agitation and aeration were maintained at 7.5, 37°C, 1000rpm

and 1vvm respectively throughout the cultivation period.

6. The overnight growth culture of yeast was transferred to fresh medium in the

fermentor.

7. The samples were collected at regular interval of 1 hr for 15 hrs and read the OD at

600nm.The OD values is tabulated.

8. The growth curve was plotted with time on X-axis and OD on Y-axis.

9. The specific growth rate and doubling time was calculated.

TABULATION

TIME

(hrs)

OD VALUE AT

600nm

RESULT

The specific growth rate and doubling time are:

µ= .

td= .

Experiment No: 2

Date :

THERMAL DEATH KINETICSAIM

To find the thermal death rate, kd using thermal death kinetics.

PRINCIPLE

A fermentation product is produced by culturing a microorganism. If contamination of

the culture occurs a number of adverse effects are in order including reduction in yield and

conversion. Hence the fermentation medium must be completely sterile before attempting to

inoculate the culture of interest.

Sterility is an absolute concept. There is no such thing as ‘partially sterile’ or almost

sterile. On a practical basis, sterility means the absence of any detectable, viable organism and

a pure culture means that only the desired organism grows in the culture vessel. We should

understand the kinetics of death. Death in this case means the failure of the cell spore to

germinate when placed in favorable condition.

Organisms are classified as (i) Thermophilic, (ii) Mesophilic, (iii) Thermophilic

depending on their heat resistant properties. The death of a particular cell is probably due to

the thermal denaturation of one or more essential proteins such as enzymes. The kinetics of

such cooperative transitions for these molecules may be either complex in time; also the rate

of the molecular process ultimately leading to the death of the cell will depend on

environmental conditions including solvent concentration.

Analysis of death rate kinetics is a first order decay for the viable population level ‘N’

is dN/dt = - kd N where kd =thermal death rate constant.

This equation yields

Nt = No e-Kd

t

Where

Nt =concentration of vegetative cells at t=0

No= concentration of viable cells at t=t

PROCEDURE:

1. 300 ml of 1.5% agar was prepared.

2. All the glass wares and the media were sterilized.

3. 100µl of the culture was inoculated in 15 ml of sterile broth.

4. Inoculated medium was heated in water bath at 85ºC.

5. 100µl of samples were drawn at an interval of 5 minutes.

6. The drawn samples were plated on a particular containing sterile agar.

7. The plated plates were incubated at 37 ºC overnight.

8. Then the grown colonies were observed.

TABULATION Temperature Time

(mins.)No.of colonies

dilution factor 10-5Nt/N0 ln(Nt/N0) Kd

Average Kd=

RESULT:

Thus the thermal death kinetics of the given bacteria was studied at 85ºC for various

time intervals. Theoretically, kd value was found to be _________ and form the graph ln (N t

/No) Vs t, kd was found to be __________.

Experiment No: 3

Date :

CELL AND ENZYME IMMOBILIZATION

AIM

To immobilize invertase enzyme and to compare the rate kinetics between both

immobilized cell and immobilized enzyme.

THEORY

The restriction of enzyme mobility on a fixed space is called immobilization. It

provides important advantage such as enzyme re-utilization and elimination enzyme recovery

and purification process. It also provides a better environment for enzyme activity product

purity is improved and efficient handling can be done in immobilization.

MATERIALS REQUIRED

4% CaCl2 solution

Yeast culture

Sodium alginate

Phosphate buffer

APPARATUS REQUIRED

Centrifuge, micropipette, glass wares.

Yeast solution:

1g of yeast was dissolved in 100ml buffer and is agitated in a shaker for 72hrs (when

the tablets are used). When yeast culture is used broth can be utilized directly.

PROCEDURE

Cell immobilization:

1) 0.4g of Sodium alginate was dissolved in 20ml of phosphate buffer. Then it was

gently heated and mixed to avoid the formation of any clumps and sodium alginate.

2) 20ml of broth culture was added directly in gelatinous form of sodium alginate.

3) The two solutions were mixed and added drop by drop to 4% CaCl2 (other

concentrations can be used) using micropipette.

4) The beads are formed and they get settled at bottom of the container.

Enzyme immobilization:

1) Sodium alginate was obtained in gelatinized form.

2) The broth culture was centrifuged at 7000rpm for 10 min in cooling centrifuge and

20ml of gelatinized sodium alginate.

3) Two solutions were mixed and added drop by drop to 4% CaCl2 (other concentrations

can be used) using micropipette.

4) The beads are formed and they get settled at bottom of the container.

Measurement of diameter of beads

1) CaCl2 was decanted and beads were washed in water.

2) They were dried with filter paper. So beads were counted and added to a measuring

flask containing water.

3) The volume displayed by 50 beads was measured. From this diameter of individual

beads was measured.

RESULT

Diameter of the bead,

1) In cell immobilization method is ___________________mm

2) In enzyme immobilization method is ______________________mm

Experiment No: 4

Date :

ESTIMATION OF KLa BY SULPHITE OXIDATION METHOD

AIM

To determine KLa, the mass transfer of coefficient of oxygen, by sulphite oxidation

method.

PRINCIPLE

KLa is the liquid phase mass transfer coefficient of oxygen in an aerated vessel. There

are many methods used in the estimation of KLa comprising physical, chemical, and biological

methods.

The sulphite oxidation technique is a chemical method to estimate KLa. It does not

require measurement of DO concentration but relies on the rate of convertion of 0.5M solution

of sodium sulphite to sodium sulphate.

It follows the given equation.

2Na2SO3 + O2 → 2Na2SO4

Na2SO4 + CaCl2 → CaSO4 + 2NaCl

The state of reaction is that as O2 enters the solution it is immediately consumed in the

oxidation of sulphite.The sodium sulphate formed,when reacted with CaCl2 forms calcium

sulphate which is obtained as a precipitate. The amount of CaSO4 obtained is equivalent to that

of sulphite oxidation rate and it is equivalent to that of O2 transfer.

The KLa is calculated from,

KLa + C* = ½ d/dt [SO4]

where C* = 7.3 mg O2/l.

MATERIALS REQUIRED

250 ml beakers

Test tubes

Pipettes

Hot air oven

Petriplate

Weighing balance

CHEMICALS REQUIRED

0.5M sodium sulphite

Calcium chloride

PROCEDURE

1. 0.5M sodium sulphite solution is prepared in aerated distilled water and purged with O2

2. Sample of 1.2 ml is collected and the solution of calcium chloride is added.

3. The precipitate is allowed to settle, calcium sulphate is washed and transferred to

preweighed dish.

4. It is dried over night in an oven at 100-1500 C.

5. Weight of CaSO4 is measured and KLa is calculated.

TABLE: 1

ESTIMATION OF KLa BY SULPHITE OXIDATION METHOD:

Time

(min)

Weight of

sulphate (g)

d[SO4] d[SO4]/dt KLa=1/2 C*

TABLE: 2

CALCULATION OF WEIGHT OF SULPHITE:

Time (t) Volume of

sulphite(ml)

Volume of

chloride (ml)

Weight of

filter paper

(W1)

Weight of

filter paper +

[SO4] [W2]

Weight of

SO4 [W2-W1]

RESULT:

Thus the KLa value from sulphite oxidation method =

Experiment No: 5

Date :

RESIDENCE TIME DISTRIBUTIONAIM

To calculate residence time distribution of fluid in the vessel for pulse input and to plot

exit age distribution.

PRINCIPLE

The mixing time denotes the time required for the tank composition to achieve a

specific level of homogeneity following the addition of the tracer pulse at a single point in the

vessel. The trace might be a salt solution, base or an acid or heated or cooled pulse. The

circulation characteristics of the vessel and the mixing time can be measured by continuously

monitoring the tracer concentration at one or several points in the vessel.

The circulation time is also important because it indicates approximately the

characteristic time interval during which a cell or a biocatalyst suspend in the agitated fluid

will circulate through different regions of the reactor ,possibly encountering different reactor

conditions along the way.

The effluent stream is a mixture of fluid elements which have resided in the reactor for

different length of time. Determination of the distribution of these residence times in the exit

stream is also a valuable indicator of the mixing and flow patterns within the vessel.

PROCEDURE

1. Two litres of distilled water was transferred into the bioreactor.

2. The agitation was maintained at 1000rpm throughout the process.

3. A pulse input of 1N potassium per magnate solution was injected with the help of

peristaltic pumps.

4. The flow rate was adjusted to maintain at a constant volume throughout the process.

5. As soon as it was injected, the zeroth hour sample was collected as spectrometric reading

was taken at 530nm.

6. Then at regular intervals of 5 mins, the sample was collected and corresponding readings

were taken until the tracer concentration turns to be negligible.

7. The standard graph was plotted for KMnO4 using the varied concentration of KMnO4

solution. (Prepared from stock containing 0.1mg/ml and its corresponding OD at 530nm)

8. The linear equation y = mx + c was obtained from the standard graph as y =

9. The output tracer concentration of each trial can then be calculated by the formula y =2.3x,

where y =OD at 530 nm.

X = con. of output tracer.

10. The difference in time is 5 min for each trial. F value can be calculated using the formula

E = c/∑c∆t

From the obtained values, E curve was plotted between E values and time and C curve was

plotted between tracer output concentration and time.

TABULATION

PREPARATION OF STANDARD GRAPH OF KMNO4

Sl.No Volume of

KMnO4

Concentration

of KMnO4

Vol of dist

water(ml)

Total

volume(ml)

OD

530nm

Time(min) OD

530nm

Tracer

output

con(c)

∆t c∆t Tc∆t E=

c/∑c∆t

RESULT

Thus the residence time distribution was estimated as _________ and E

curve was plotted.

Experiment No: 6

Date :

DETERMINATION OF OVERALL HEAT TRANSFER COEFFICIENT

AIM

To determine the overall heat transfer coefficient ‘µ ‘ for a bioreactor.

PRINCIPLE

Driving force for heat transfer is the temperature difference between any two

bodies.Two bodies at different temperature, when they are in constant tend to attain the same

temperature by heat transfer. This phenomenon is used in reactors to heat or cool the medium

to desired temperature. To determine the flow rate, temperature of the heating or cooling is

performed effectively and efficiently. It is necessary to know the overall heat transfer

coefficient ‘ µ ‘. The transferred heat is proportional to the temperature gradient and inversely

proportional to the area, hence

Q α A∆T

Q = UA∆T

Thus by knowing the overall rate of heat transfer (Q) we can determine µ by a

simple thermodynamic relation.

Q = mF Cp [Tₒ-Ti]

Where

mF = mass flow rate of heating or cooling substance (kg/m.s)

Cp = specific heat capacity of heat substance (J/kg.K)

To= outlet temperature of substance for heating or cooling( K)

Ti = inlet temperature of substance for heating or cooling (K)

U=mFCp (To-Ti)

A∆T

∆T can be defined in many ways and the best would possibly be logarithmic mean temperature

difference (LMTD).

LMTD= Ti-To

ln [TR-To]

[TR-Ti]

Where TR- Reactor temperature

Also the value of U depends on the nature of material across the heat is being

transferred, the system geometry, the nature of fluid etc.The dependence of 'U' on all of the

above is quiet complex and is evaluated using emperically.

1 = 1 + 1 × do + xw × do Vo ho hi di kw di

where ho-heat transfer coefficient of liquid (film)outside

hi-heat transfer coefficient of liquid (film) inside

PROCEDURE

1. The reactor was filled with water.

2. The heater inside the reactor was switched on and the reactor temperature was

noted.

3. To maintain the required temperature, cold water was passed into the reactor

and its temperature was noted.

4. The reactor was allowed to agitate at 500rpm and was left running until steady

state was obtained.

5. Now the reactor temperature and outlet water temperature was noted. The flow

rate of the outlet water being taken at different flow condition.

TABULATION

RPM Time(sec) Temperature(0C)

Ti To TR

RESULT

The overall heat transfer coefficient was found to be

(i)At 800rpm (flowrate of kg/s) = .

(ii)At 800rpm (flowrate of kg/s) = .

Experiment No: 7

Date :

ESTIMATION OF KLA BY DYNAMIC GASSING OUT METHODAIM

To estimate the volumetric mass transfer coefficient KLa by dynamic degassing method.

PRINCIPLE

The estimation of KLa of a fermentation system by gassing out technique depends on

monitoring the increase in dissolved oxygen concentration during aeration and agitation. The oxygen

transfer rate will increase during the period of aeration as CL approaches c* due to decline in the

driving force (c*- cL). The oxygen transfer rate at any time will be equal to the slope of the tangent to

the curve of values of dissolved oxygen concentration against the time of aeration.

To monitor the increase in DO over an adequate range, it is necessary first to decrease the

oxygen level to a low value. Two methods have been employed to achieve this lowering of the DO

concentration the statistical method and dynamic method.

In dynamic method the respiratory activity of a growing culture is used in the fermentor to

lower the oxygen level prior to aeration. Therefore the estimation has the advantage of being carried

out during fermentation.

PROCEDURE

1. The two litre fermentor was aerated by the supply of air from tanks through a compressor.

2. Air was passed through regulated valve system.

3. It is introduced at the bottom of the filter through a sparger.

4. The air supplied to the fermenter was stopped by shutting of the regulating valve. The fall in the

DO concentration was noted down at regular time intervals.

5. After the DO level reaches 58% the valve was opened to allow the inflow of air.

6. The rise in the DO concentration was noted regular intervals till the value becomes constant.

7. A graph was plotted by taking time in X axis and DO in Y axis.

TABULATION

Degassing:

Time(s) D.O (%) QO2

Gassing:

Time(s) D.O(%) dCL/dt=(c*-cL)ΔT dCL/dt + QO2

RESULT

The value of KLa was calculated at different intervals by dynamic degassing procedure.

The KLa was found to be

Experiment No: 8

Date :

FED BATCH REACTOR

AIM

To run the fed batch reactor for the production of alkaline protease.

PRINCIPLE

Fed batch cultivation is carried out in order to overcome substrate inhibition. Here the

substrate is given as a small amount at various times. The term fed batch is used to describe

batch cultures which are fed continuously or sequentially with the medium, without the

removal of culture fluid.

In the fed batch culture nutrients are continuously or semi continuously fed while

effluent is removed such a system is called repeated fed batch culture. The fed batch culture is

usually used to overcome substrate inhibition or catabolic repression by intermittent feeding of

substrate. It improves the productivity of the fermentation by maintaining low substrate

concentration.

Fed batch operation is also called semi continuous system or variable volume

continuous culture.

PROCEDURE

1. 2litre of 2X LB Medium was prepared.

2. The medium was poured in the fermentor and sterilized.

3. After sterilization, 100ml of pre inoculum with OD 0.9 is inoculated.

4. The pH is 7.1, temperature is 37o C and aeration is maintained at constant.

5. Zero hour sample was withdrawn immediately after inoculation.

6. Samples are collected for every hour and read at 600nm.

7. The remaining samples are centrifuged at 1000rpm for 10minutes.

8. The pellet is processed to obtain the product.

TABULATION

Time(hr) O.D(600 nm)

RESULT

A graph was plotted between the time and the cell growth (OD) to study the growth

of the cells in culture.

Experiment No: 9

Date :

ESTIMATION OF KLa BY POWER CORRELATION METHOD

AIM

To calculate KLa by power correlation method.

PRINCIPLE

A widely used correlation for stirred vessels relates KLa directly to gas velocity and

power input to the stirrer. All the effects of flow and turbulence on bubble dispersion and the

mass transfer boundary layer are represented by the power term. An expression for stirred

fermentation containing non-viscous media is

KLa = 2 X 10 -3(P/V) 0.7 UG0.2

KLa is the combined mass transfer co-efficient in units of S-1.P is the power dissipated

by the stirrer in W and V is the fluid volume in m3.UG is the superficial gas velocity in ms-

1.Superficial gas velocity is defined as the volumetric gas flow rate divided by the cross

sectional area of the fermenter.

Application of this correlation to the production vessels upto 25 m3 in volume has been

found to overestimate rate of O2 transfer by about 100%. This correlation does not depend

upon the sparger or stirrer design. The power dissipated by the impeller determines KLa

independent of stirrer type. KLa can be determined by raising the superficial gas velocity in the

reactor.

MATERIALS REQUIRED

Fermentor

Sterile distilled water

PROCEDURE

1. The impeller diameter and the speed of the impeller are measured.

2. NRe is measured from the given formula.

3. Then for turbulent flow the power number is measured from the power

characteristic of mixing impeller.

4. Then the power is measured form the following formula

P=Np ρN3Di 5

5. The inner diameter of the fermenter is measured. Then the superficial velocity is

calculated from the volumetric flow rate and cross sectional area of the fermenter.

6. By knowing the power and volume of the fermenter KLa can be calculated form the

formula.

RESULT

The value of KLa calculated by power correlation method =

INFERENCE

The KLa value calculated bypower correlation method gives the O2 transfer coefficient based

on the power required to run the reactor and dimension of the vessel.

Experiment No: 10Date :

DETERMINATION OF POWER REQUIREMENT FOR BATCH REACTOR

AIM

To measure the power required to operate a batch reactor.

PRINCIPLE

Usually electrical power is required to drive impeller for a given stirrer vessel for a

given speed the power required depends on the resistance offered by the fluid to the rotation of

the impeller mixing power for non aerated fluid depends on the stirrer speed, the impeller

diameter and geometry and the properties of fluid such as viscosity and density. The

relationship between the variables is usually expressed in terms of diamensionless number

such as impeller Reynolds number NRe ant the power number Np.

Power number is defined as:

Np= P/ρ Ni3 Di5

Thus the power number is the ratio of external force exerted to the internal force imparted to

the liquid. For a given impeller the given relationship between Np Vs NRe depends on flow

regim in the tank.

MATERIALS REQUIRED

Fermenter, sterile deionized water

PROCEDURE

The impeller diameter, 48mm was obtained from the equipment manuel and the speed

of the impeller 1000rpm was noted. From speed controller the NRe was calculated using

viscosity and density of water which is 1000Kg/m3 and 1X 10-3 Kg/msec respectively.

NRe = Di2 Ni ρ/ µ

From the value of NRe = 38407 the characteristic of the flow of mixing by russian turbine was

found to be turbulent the power number was obtained from the Np Vs NRe curve as shown.

The power number NRe for turbulent region for Rushton turbine and using the power number

value the power required was calculated using the formulae

P= Np ρ Ni3Di5

RESULT

Thus the power required to run a batch reactor was found to be P =

Experiment No: 11

Date :

EFFECT OF TEMPERATURE ON AMYLASE ACTIVITY

AIMTo determine the effect of temperature on the activity of amylase.

PRINCIPLE

Amylase is a hydrolytic enzyme which breaks down many polysaccharides like starch

which is a polymer of glucose linked by α-1, 4 glycosidic bonds to yield a disaccharide maltose

as an end product. α-amylase is also called as α-1,4 gluconohydroxylase.

Amylase

(C6H12O6 )n + nH2O n(C12H22O11)

starch maltose

Saliva is a good source of amylase. The activity of enzyme depends on the temperature

at which reaction is carried out. The optimal temperature required for the activity of amylase

can be calculated by this experiment.

The rate of reaction is monitored by measuring the amount of substrate consumed or by

measuring the product formed. Here the substrate is starch and the product is maltose. Since

both are colorless DNS is used.DNS when added with maltose and heated it reduces to 3-

amino, 5 nitrosalicylic acid is determined by the color change in this solution. The conversion

of DNS to 3-amino, 5 nitrosalicylic acid varies based on maltose concentration. It gives pure

orange red for high concentration of maltose and shows very less color change by low

concentration. The color change is noted spectrophotmetrically at 520nm. The concentration of

maltose formed can be calculated from the absorbance.

MATERIALS REQUIRED

Glasswares

Beaker, standard flask, measuring cylinder, pipettes, test tubes.

Equipments

Weighing balance, magnetic stirrer ,spectrophotometer .

Chemicals

DNA, starch, Sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium

hydroxide, sodium chloride, distilled water, α-amylase.

PROCEDURE

1. The required number of test tubes were taken, washed, dried and labeled.

2. Different reagents were added following the table.

3. Immediately after addition of enzyme, add 0.5 ml of 2N NaOH to all tubes

except zero time control.

4. The tubes were incubated at 37ºC for 10 minutes.

5. After incubation, the reaction was stopped by adding 0.5 ml of 2N NaOH to all

tubes except zero time control.

6. 0.5 ml of DNS reagent was added to each of the tubes and was vortexed and

heated in boiling water bath for 10 minutes.

7. The test tubes were cooled to room temperature and absorbance read at 520 nm

8. The activity of enzyme was calculated by the formula.

Test OD- Control OD

Enzyme Activity = Reaction time

TABULATION

TempºC

Buffer

Ml

Starch

ml

NaCl

Ml

Mix

wel

l and

incu

bate

for

10 m

in

Amylase

ml

Mix

wel

l and

incu

bate

for

10 m

in

NaOH

ml

M

ix w

ell a

nd in

cuba

te fo

r 10

min

DNS

ml

D.water ml

K

eep

in b

oilin

g w

ater

bat

h fo

r 10

min

OD

37T 2.5 2.5 1 0.5 0.5 0.5 -

C 2.5 2.5 1 0.5 - 0.5 -

B 2.5 2.5 1 - 0.5 0.5 0.5

50T 2.5 2.5 1 0.5 0.5 0.5 -

C 2.5 2.5 1 0.5 - 0.5 -

B 2.5 2.5 1 - 0.5 0.5 0.5

70T 2.5 2.5 1 0.5 0.5 0.5 -

C 2.5 2.5 1 0.5 - 0.5 -

B 2.5 2.5 1 - 0.5 0.5 0.5

80T 2.5 2.5 1 0.5 0.5 0.5 -

C 2.5 2.5 1 0.5 - 0.5 -

B 2.5 2.5 1 - 0.5 0.5 0.5

OBSERVATION

The graph plotted between temperature and enzyme activity decreases as the

temperature increases.

Enzymes are proteinaceous substance. Denaturation begins to occur at 40-50oC. one

physical mechanism for the phenomenon is obvious as the temperature increases, the atom in

the enzyme molecule have greater tendency to move. Eventually the aquire sufficient energy

to overcome the weak interaction holding the globular structure together and deactivation

follows. This makes the enzyme to lose its activity at higher temperature.

RESULT

The effect of enzyme activity at different temperature was determined. Maximum enzyme

activity was observed at ________.

Experiment No: 12

Date :

EFFECT OF pH ON AMYLASE ACTIVITYAIM

To determine the effect of pH on the activity of amylase.

PRINCIPLE


which is a polymer of glucose linked by α-1, 4 glycosidic bond to yield a disaccharide maltose


Amylase

(C6H12O6 )n + nH2O n(C12H22O11)

starch maltose

Saliva is a good source of amylase. The activity of enzyme depends on the pH at which

reaction is carried out. The optimal pH required for the activity of amylase can be calculated by

this experiment.





of DNS to 3-amino,5 nitrosalicylic acid varies based on maltose concentration. It gives pure




MATERIALS REQUIRED

Glasswares

Beaker, standard flask, measuring cylinder, pipettes, test tubes .

Equipments

pH meter, weighing balance, magnetic stirrer, spectrophotometer .

Chemicals

DNA ,starch ,Sodium dihydrogen phosphate ,disodium hydrogen phosphate, sodium hydroxide,

sodium chloride, distilled water, α-amylase.

PROCEDURE

1) The required number of test tubes were taken, washed, dried and labeled.

2) Different reagents were added following the table.

3) Immediately after addition of enzyme, add 0.5 ml of 2N NaOH to all tubes except zero

time control.

4) The tubes were incubated at 37ºC for 10 minutes.

5) After incubation, the reaction was stopped by adding 0.5 ml of 2N NaOH to all tubes


6) 0.5 ml of DNS reagent was added to each of the tubes and was vortexed and heated in

boiling water bath for 10 minutes.

7) The test tubes were cooled to room temperature and absorbance read at 520 nm

8) The activity of enzyme was calculated by the formula.

Test OD- Control OD


TABULATION

EFFECT OF pH ON AMYLASE ACTIVITY

pH Buffer

ml

Starc

h

ml

NaC

l

Ml

Mix

wel

l and

incu

bate

at Amyla

se

ml

M

ix w

ell a

nd in

cuba

te a

t

370 C

for

10 m

in

NaO

H

ml

M

ix w

ell a

nd in

cuba

te a

t DNS

ml

D.wat

er

ml

M

ix w

ell a

nd in

cuba

te a

t OD

4

T 2.5 2.5 1 1 0.5 0.5 -

C 2.5 2.5 1 1 - 0.5 -

B 2.5 2.5 1 Nil 0.5 0.5 1

370 C

for

10 m

in

370 C

for

10 m

in

370 C

for

10 m

in

7

T 2.5 2.5 1 1 0.5 0.5 -

C 2.5 2.5 1 1 - 0.5 -

B 2.5 2.5 1 Nil 0.5 0.5 1

9

T 2.5 2.5 1 1 0.5 0.5 -

C 2.5 2.5 1 1 - 0.5 -

B 2.5 2.5 1 Nil 0.5 0.5 1

OBSERVATION

Thus, the pH of the mixture varies the activity of the enzyme absorbance at a range of

pH 5-8. The activity versus pH shows an inverted V-curve, which means the activity is

maximum at pH 7.0.

RESULT

The amylase activity at different pH was determined. Maximum amylase activity was

observed at _______.

Experiment No: 13

Date :

DETERMINATION OF ENZYME KINETICS PARAMTERS BY VARYING SUBSTRATE CONCENTRATIONS

AIM

To determine the activity of amylase enzyme at different substrate concentrations

using starch as substrate.

PRINCIPLE


which is a polymer of glucose linked by α-1, 4 glycosidic bonds to yield a disaccharide maltose


Amylase

(C6H12O6 )n + nH2O n(C12H22O11)

starch maltose

Saliva is a good source of amylase. The activity of enzyme depends on the temperature

at which reaction is carried out. The optimal temperature required for the activity of amylase

can be calculated by this experiment.





of DNS to 3-amino,5 nitrosalicylic acid varies based on maltose concentration. It gives pure




MATERIALS REQUIRED

Glasswares

Beaker, standard flask, measuring cylinder, pipettes, test tubes.

Equipments

pH meter, weighing balance, magnetic stirrer cum hot

plate ,spectrophotometer ,micropipette.

Chemicals

Sodium potassium tartarate ,dinitrosalicylic acid (DNS) ,starch ,Sodium dihydrogen

phosphate, disodium hydrogen phosphate, glass, sodium chloride, distilled water, α-amylase,

glass. .

PROCEDURE

1) The required number of test tubes were taken, washed, dried and labeled.

2) Different reagents were added following the table.

3) Immediately after addition of enzyme, add 0.5 ml of 2N NaOH to all tubes except zero

time control to stop the reaction .

4) The tubes were incubated at 37ºC for 10 minutes.

5) After incubation, the reaction was stopped by adding 0.5 ml of 2N NaOH to all tubes


6) 0.5 ml of DNS reagent was added to each of the tubes and was vortexed and heated in

boiling water bath for 10 minutes.

7) The test tubes were cooled to room temperature and absorbance read at 520 nm.

8) The activity of enzyme was calculated by the formula,

Test OD- Control OD


TABULATION

DETERMINATION OF ENZYME KINETICS PARAMTERS BY VARYING

SUBSTRATE CONCENTRATIONS

Starch Starch

ml

NaCl

ml

M

ix w

ell a

nd in

cuba

te a

t 370 C

for

10 m

in

Amylase

ml

Mix

wel

l and

incu

bate

at 3

70 C fo

r 10

min

NaOH

ml

Mix

wel

l and

incu

bate

at 3

70 C fo

r 5

min

DNs

ml

D.water

ml

Kee

p in

boi

ling

wat

er b

ath

for

10 m

in

OD

2

T 2.5 1 0.5 0.5 0.5 -

C 2.5 1 0.5 - 0.5 -

B 2.5 1 - 0.5 0.5 0.5

4

T 2.5 1 0.5 0.5 0.5 -

C 2.5 1 0.5 - 0.5 -

B 2.5 1 - 0.5 0.5 0.5

6

T 2.5 1 0.5 0.5 0.5 -

C 2.5 1 0.5 - 0.5 -

B 2.5 1 - 0.5 0.5 0.5

8

T 2.5 1 0.5 0.5 0.5 -

C 2.5 1 0.5 - 0.5 -

B 2.5 1 - 0.5 0.5 0.5

10

T 2.5 1 0.5 0.5 0.5 -

C 2.5 1 0.5 - 0.5 -

B 2.5 1 - 0.5 0.5 0.5

S 1/S V 1/V

RESULT

The maximum amylase activity is observed as _________ with increase in substrate

concentration.

Experiment No: 14

Date :

ION EXCHANGE CHROMATOGRAPHYINTRODUCTION

Chromatography is used to separate organic compounds on the basis of their charge,

size, shape and their solubility. A chromatography consists of a mobile phase and a stationary

phase. In ion exchange chromatography separation is based on charge of the molecule.

Proteins contain many ionizable groups on the side chains of their amino acids as well as their

amino and carboxyl- termini. DEAE cellulose is a weak anion exchanger;it will bind to the

opposite charge of the protein of interest. This experiment demonstrates the purification of

Immunoglobulin G(Ig G) using Ion exchange chromatography technique and estimates the

protein concentration and checks the quantity with specific antibody by immunodiffusion

method.

PRINCIPLE

In this experiment, DEAE cellulose (weak anion-exchanger) is used to purify the

Immunoglobulin G. Purification of Ig G is based on the interaction between positively charged

DEAE groups and the negatively charged proteins of interest. A crude sample is added to the

column; everything passes through except protein of interest. Unbound proteins completely

removed by washing. Immunoglobulin G is eluted with elution buffer by changing the pH.

DEAE cellulose gradually loses the charge at higher PH value. Quantity of the Ig G can be

determined by SDS – PAGE method by comparing the results before and after purification.

REAGENTS REQUIRED

S.No Name of the reagent Quantity Storage condition

1. 10X Equilibration

buffer

30 ml 4°c

2. 10x Washing buffer 30ml 4°c

3. 5X Elution buffer 20ml 4°c

4. Crude sample 5ml -20°c

5. Anti bovine Ig G 100µl -20°c

6. DEAE –celulose 5ml 4°C

7. Agarose 300mg RT

8. Normal saline 60 ml RT

9. Column 1 No RT

MATERIALS REQUIRED

Colorimeter or spectrophotometer

Glass cuvette, Glass slides, Gel punch and template.

Microfuge tubes, micropipette and tips.

Note:

Thoroughly mix the DEAE cellulose (ligand) before packing.

Avoid air bubble formation while packing the gel into the column.

Do not allow the column gets dried.

Discard the ligand after each use and wash with the distilled water.

Working solution preparation:

Equilibration buffer (10X)

Take one volume of 10X solution and add nine volumes of distilled water, it gives 1X

equilibration buffer.

Washing buffer (10X)

Take one volume of 10 X solution and add nine volumes of distilled water, it gives 1X

washing buffer.

Elution buffer (5 X):

Take one volume of 5x solution and add four volumes of double distilled water.

It gives 1X solution buffer.

PROCEDURE

1. Take one volumes of DEAE –cellulose suspension to a chromatography column

and allow it to settle.

2. Remove the bottom cap and add 20ml 1X equilibration buffer and let it pass

through the columns, replace the bottom cap.

3. Gently add 1ml of crude sample into the column replace the top cap and incubate

at room temperature with mixing often.

4. After incubation period, allow the gel to settle.

5. Allow drain off the sample completely

6. Wash the column with 20ml of 1x washing buffer .Allow the wash buffer to drain

off completely and discard.

7. Add 5ml of elution buffer to elute to elute bound Ig G . collect the fraction as 1ml

in a sterile microfuge tube.

8. Read the absorbance value of 280nm and mix the fractions having A280 above 0.2.

CALCULATION OF IMMUNOGLOBULIN –G CONCENTRATION

Applying the standard formula to calculate the immunoglobulin –G concentration.

A280 sample absorbance ×0.76×dilution factor

At A280 an absorbance of 1.0 (1cm cuvette) is equivalent to a bovine Ig G concentration of

0.76mg/ml

RESULT

Thus the relative centrifugal force was determined for different rpm values of the

centrifuge and it was found to be __________.

Experiment No: 15

Date :

CENTRIFUGATION

AIM

T o determine the relative centrifugal force generated by a given centrifuge.

PRINCIPLE

The particles can be separated from liquid by application of centrifugal force. This

method increases force on particle. The high settling force means that particle rate of settling

can be obtained with much smaller particle size than in gravity settler. This high centrifugal

force doesn’t change the relative settling of particles but overcome disturbances caused by

Brownian movement and free convention current.

This principle involved includes the whirling of particles about an axis as centre point

at a constant radial distance from particles acted by force. The centrifugal force act in a

direction towards centre of rotation. The particles that are being rotated in this cylinder also

exerts on equal and opposite force. This force cause settling. The equation for calculation of

centrifugal force is

a = γw2

thus centrifugal force , Fc = m γw2

Fc =mv2/γ

N =60v/2πr

Thus Fc =mγ(2πN/60)2

Fc =0.000341 m γN2

Now,

Gravitational force =mg

Therefore,

Fc/Fg = 0.0081118γN2

MATERIALS REQUIRED

5ML Pipette

Centrifuge tube

Centrifuge

Cell culture

PROCEDURE

1. 5ml of yeast culture was taken in a centrifuge tube.

2. Initially sample was subjected to centrifugation at 5000rpm for 3 minute at room

temperature.

3. The rpm and time were altered until a clean fluid was obtained.

4. The same procedure was repeated for different rpm values.

OBSERVATION

When rpm value was increased the relative centrifugal force of the centrifuge also

increases.This shows that RCF of the centrifuge is directly proportional to the rpm value.

RESULT

Experiment No: 16

Date :

ALKALINE LYSIS

AIM

Alkaline lysis is a method used to break cells, open to isolate plasmid DNA or other

cell components such as proteins. Alkali acts on the cell wall in a number of ways including

saponification of lipid. Bacteria containing plasmid of interest is first grown, and then lysed

with a strong alkaline buffer consisting of a detergent sodium dodecylsulphate (SDS) and a

strong base sodium hydroxide. The detergent breakes the membrane phospholipid bilayer and

the alkali denature the protein involved in maintaing the structure of cell membrane. Through

a series of step involving agitation, precipitation, centrifugation and removal of supernatant,

cellular debris is removed and the plasmid is isolated and purified

Also, alkaline lysis sometimes is used to extract plant genetic material. The plant cells

are subjected to a strongly alkaline solution containing a detergent usually a zwitterionic or

nonionic detergent such as Tween 20, and the mixture is incubated at high temperature. This

method is not used in often due to the tendency of sodium hydroxide to damage genetic

material, reducing DNA fragment size

MATERIALS REQUIRED

Plasmid, alkali, SDS, Potassium acetate, Isopropanol, TE buffer

PROCEDURE

1. Plasmid or cosmid containing E.coli cells are grown and lysed with alkali.

2. The cell debris and chromosomal DNA is precipitated with SDS and potassium

acetate.

3. After pelleting the debris the plasmid or the cosmid DNA is precipitated from the

supernatant with isopropanol and the DNA resuspended in TE.

4. The expected yield from a 5ml culture is 5-10µg for plasmid and 2-5µg for

cosmids. The procedure can be scaled up many folds.

RESULT

Thus the alkaline lysis method was done to isolate plasmid DNA

Experiment No: 17

Date :

ULTRASONICATIONAIM

To study the ultrasonication technique and to calculate the difference in OD value

before and after sonication.

PRINCIPLE

Disruption of cell is an important stage in the isolation and preparation of intracellular

products. Single cell organisms consist of semi permeable tough, rigid, outer cell wall

surrounding the protoplasmic membrane and cytoplasm. The energy applied must be great

enough to break the cell membrane or cell wall, yet it should be gentle enough .Microbes

differ greatly in their sensitivity to ultrasonic processing will typically cause temperature of

the sample to increase especially with small volume. Since high temperature inhibits

cavitations, the sample temperature must be kept as low as possible, preferably just above its

freezing point temperature elevation can also be minimized by using the phase.

The ultrasonic power supply converts 50/60 Hz line voltage to a high frequency

electrical energy, which is transmitted to piezoelectric transducer within the converter, where

it converted to mechanical vibrations. The vibrations from the converter are intensified by the

probe creating pressure waves in the liquids. This action forms millions of microscopic

bubbles which expand during negative pressure. As the bubbles implode, they cause millions

of shock waves and eddies to radiate outwardly from the side of collapse as well as generate

extreme pressure and temperature at the implosion site and this phenomenon is called

cavitations.

MATERIALS REQUIRED

5) Bacillus subtilus culture

6) Phosphate buffer

7) Centrifuge tubes

8) Ultrasonicator

9) UV spectrophotometer

PROCEDURE

1. 5ml of culture was taken in 8.5ml tube and it was centrifuged. After centrifugation the

supernatant is discarded.

2. The pellet was suspended in 10mM phosphate buffer

3. The suspension was again centrifuged and supernatant was collected.

4. The absorbance was measured at 280nm.

5. The cells were subjected to sonication.

6. The power, pulse, amplitude and time were set for sonication.

7. The sonicated sample was centrifuged and absorbance value was read at 280nm.

TABULATION

Before sonication OD value at

280nm

After sonication OD value at

280nm

Difference

OBSERVATION

The sonication was done and OD values were taken before and after sonication were

performed.

INFERENCE

Comparing the OD values, we can infer that some of the intracellular products were released

to the surroundings

RESULT

The ultrasonication was performed and thus difference in OD value before and after sonication

was found to be ___________.

Advanced Bioprocess and Downstream-III Sem PG

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