A.C COLLEGE OF TECHNOLOGY ANNA UNIVERSITY, CHENNAI-600025.

BIOPROCESS LAB RECORD

NAME ROLL NO

: :

DEPARTMENT : IBT

BONAFIDE CERTIFICATE A.C.COLLEGE OF TECHNOLOGY

NAME :

BRANCH

ROLL NO.

This is to certify that _____________________________________ of _____________________________________ has completed the BIOPROCESS LAB-I (IB9306) in the year 20112012 Submitted for the practical examination conducted on ________________________

INTERNAL EXAMINER

EXTERNAL EXAMINER

INDEX

S NO
1 2 3 4 5 6 7 8 9 10 11 12

DATE

TOPIC

Exp No: Date : STUDY OF ENZYME KINETICS OF INVERTASE

Aim: To determine the enzyme kinetic parameters Vmax, Km and kcat of the enzyme invertase. Theory: The hydrolysis ('inversion') of sucrose, completely or partially, to glucose and fructose provides sweet syrups that are more stable than pure sucrose syrups. Sucrose is -Dglucopyranosyl (1,2)--D-fructofuranoside and can therefore be hydrolysed both by glucosidases and -fructofuranosidases. Invertase is a -fructofuranosidase that hydrolyses sucrose as well as other b-fructans such as raffinose.

Yeast (Saccharomyces cerevisiae) invertase is essentially an intracellular enzyme and disruption of cell membranes is necessary for its extraction. A crude extract prepared by autolysis of dried baker's yeast gives satisfactory activity with no interfering activities. This practical exercise demonstrates how the kinetic parameters Km and Vmax may be determined for this industrially important enzyme. The kinetic parameters are studied by plotting the Michaelis-Menten curve and Lineweaver Burke Plot. The Michaelis Menten equation relating reaction rate and concentration of substrate is given by

Lineweaver-Burke equation is given by

Materials required: Invertase : 1 ml stock is diluted with 100ml phosphate buffer Sucrose solution 50 mM Phosphate Buffer (pH= 7.0,Potassium) 0.05M Procedure: 1. Sucrose was taken in increasing volumes of 0, 0.5, 1.0, 1.5, 2.0, 2.5& 3.0ml in different test tubes. 2. The volume was made upto 3ml using phosphate buffer (pH= 7.0). 3. 200 l of invertase is added was added to the test tubes and incubated for 20 minutes at room temperature. 4. The reaction mixture is boiled at 90 C for 5 minutes to stop the reaction. 5 The velocity of the product formation, the glucose was analysed by using GOD- POD kit by taking 1.8 ml GOD POD reagent and 0.2 ml of the reaction mixture and read at 540 nm.

The readings are tabulated and the kinetic parameters are calculated.

Table 1: Standard values of glucose Glucose stock 0.2 mg/ml

Glucose Volume l 20 40 60 80 100 4 8 12 16 20 Quantity g

Absorbance OD @ 540 nm

Table 2: Tabular column for the calculation of velocity of the reaction for various substrate concentration

S.no sucrose

1/S glucose

1/V

Vol of Sucrose

Molarity of Sucrose in 3ml (S)

OD @ Conc. of glucose 540 nm

Velocity of reaction (V)

(ml)

mM

g/l

mM

mM/min

Graphs to be plotted: 1. Michaelis Menten plot (V vs. S). 2. Lineweaver Burk plot (1/V vs. 1/S). Results and Inferences:

Exp. No: Date: EFFECT OF TEMPERATURE ON ENZYME ACTIVITY Aim: 1. To study the effect of change in temperature on enzyme activity and calculate and A. Theory: The temperture of a system is to some extent a measure of the kinetic energy of the molecules in the system. Increases in the temperature of a system results from increases in the kinetic energy of the system. This has several effects on the rates of reactions. 1) More energetic collisions 2) The number of collisions per unit time will increase. 3) The heat of the molecules in the system will increase. Given the above considerations, each enzyme has a temperatuare range in which a maximal rate of reaction is achieved. This maximum is known as the temperature optimum of the enzyme. Invertase (systematic name: beta-fructofuranosidase) is an enzyme that catalyze the hydrolysis (breakdown) of sucrose. Invertase, usually derived from yeast, has an Optimum temperature of 50C and an optimum pH of 4.5. A further increase in temperature will cause the activity to decline, and with further increase in temperature the activity will drop to nil. It is because at higher temperature the 3-D structure of the enzyme is destabilized and may even collapse resulting in its total denaturation. When the 3-D structure of the enzyme is destabilized the geometry of the active site is also affected resulting in slowing down and even total abolition of catalytic activity. Materials required: Invertase prepared by sonication (French press) of Bakers yeast solution (1% suspended in phosphate buffer of pH 7 is the stock solution. Invertase : 1 ml stock is diluted with 100ml phosphate buffer Sucrose solution: 50 mM 0.05M Phosphate buffer at pH of 7.0 Ea

Procedure: Take 5 labelled test tubes. 3ml of 50 mM sucrose solution was added to each of the tubes. 200 l of the invertase solution of was added to each of the test tubes, excluding the blank.

Incubated the test tubes for20 minutes, one each at different temperatures -- 30C, 40C, 50C and 70C. The reaction was stopped by heating the test tubes for 5 min in a water bath kept at 90C. Glucose, the product formed was analysed using the GOD-POD kit. The readings were tabulated and Vmax is calculated.

Observation:

Table 1: Standard values of glucose Glucose stock: 0.2 mg/ml Glucose Volume l Quantity g Absorbance OD at 540 nm

Table 2: Tabular column for the Effect of Temperature on Enzyme Kinetics Amount of Sucrose (ml) Amount of Phosphate Buffer (ml) Amount of enzyme (l) Temperature of incubation (oC) for 20 mins Absorbance at 540nm

Table 3: Tabular column for the Velocity of Reaction Concentration of Glucose (g/ml) Velocity of Reaction (V) mM/min (x10-3) Temperature (oC)

Table 4: Tabular column for the Arrhenius Relations Temperature (oC) 1/T Velocity (x10-3) K ln K

Graphs to be plotted: 1. Temperature profile for Invertase (Vmax vs. Temperature) 2. Plot graph of lnK vs. 1/T and calculate Ea and A. Result:

Exp No: Date : EFFECT OF PH ON ENZYME KINETICS

Aim: To study the effect of change in pH on invertase activity. Theory: Enzymes have ionic group in the active site and these groups must be in a suitable form (acid or base)n to function. Variation in pH of the medium results in changes in the ionic form of the active sites and changes in the activity of the enzyme and also in the reaction rate. Changes in pH may also alter the 3D shape of the enzyme. For this reason, enzyme is active in a certain pH range. The pH may affect the maximum reaction rate and stability of enzyme. If the substrate contains ionic group, then pH of the medium affects the affinity of substrate to enzyme. At extreme pH tertiary structure of protein may be disrupted and protein may be denatured. At moderate pH values, tertiary structure is not disrupted. Change in pH affects shape and active site of enzyme, so substrate binding is hindered and affects process catalysis.

Materials required: Invertase prepared by sonication (French press) of Bakers yeast solution (1% suspended in acetate/phosphate buffer of appropriate pH - 4, 5, 6, 7, 8) is the stock solution. Invertase : 1 ml stock is diluted with 100ml of appropriate buffer Sucrose solution 50 mM made in buffer of appropriate pH (4, 5, 6, 7, 8) Buffers of pH 4, 5, 6, 7, 8 prepared using appropriate buffers of 0.05M. Procedure: 1. Take 6 labelled test tubes 2. 3ml of 50 mM sucrose solution of varying pH (4 to 8) was added to each of the 5 tubes. To one test tube (Blank), add 50 mM sucrose of pH 7. 3. 200 l of the invertase solution of appropriate pH was added to each of the test tubes, excluding the blank. 4. Incubated for 20 minutes at room temperature. 5. The reaction was stopped by boiling the test tubes for 5 min in a water bath. 6. Glucose, the product formed was analysed using the GOD-POD kit.

Observation: Table 1: Standard values for glucose concentration Stock: Amount of glucose (mg/ml) OD @ 540 nm

Table 2: Tabular column for the calculation of Vmax

S.no

pH

OD @ 540 nm

Vmax

Graphs to be plotted: pH profile for invertase (Vmax vs. pH) Result:

Exp no: Date:

Enzyme Inhibition Kinetics

Aim: To study the inhibition kinetics of CuSO4 on Enzyme Invertase. Theory: Inhibitors are substances which tend to decrease the rate of an enzyme catalyzed reaction. Reversible inhibitors bind to an enzyme in a reversible fashion and can be removed by dialysis whereas irreversible inhibitors cannot be removed. There are different types of reversible inhibitors: i) Competitive inhibitors: These inhibitors closely resemble the substrates whose reactions they inhibit. They compete for and bind to the same active site on the enzyme forming a dead end [SI] complex. ii) Uncompetitive inhibitors: These inhibitors bind to the enzyme substrate complex and not to the free enzyme. [ESI] is formed which is a dead end complex. iii) Mixed inhibitors: These inhibitors bind to either the substrate binding site or the enzyme-substrate complex, thereby, inhibiting product formation.

Materials Required: Invertase prepared by sonication (French press) of Bakers yeast solution (1% suspended in citrate buffer of pH 4.5 is the stock solution. Invertase: 1 ml stock is diluted with 100ml citrate buffer Sucrose solution: 50 mM 0.05M citrate buffer at pH of 4.5

Procedure: Sucrose solution was taken in increasing volumes of 0.5, 1, 1.5, 2.0, 2.5 and 3ml in different test tubes and is done in duplicates. Volume is made upto 3 ml in the test tubes with the buffer 300 l of 0.1 M CuSO4 solution was added to one set and to the other 300l of buffer was added. The reaction was started by adding 200 l invertase to each test tube. The reaction mixture was incubated for 20 minutes at room temperature. The reaction was stopped by boiling the tubes for 5 minutes. Glucose, the product formed was estimated using the GOD-POD kit. The readings were tabulated.

Observation: Table 1: Standard values of glucose Glucose stock: 0.2 mg/ml Glucose Volume l Quantity g Absorbance OD @ 540 nm

Table 2: Tabular column for the Enzyme activity with inhibitor

Amount of Sucrose (ml)

Amount of Phosphate buffer (ml)

Amount of 0.1M CuSO4 (l)

Amount of Enzyme (l)

OD at 540nm

Table 3: Tabular column for the Line-weaver Burke graph for reaction with inhibitor

[S] mM

[P] mM

Tine (min)

Velocity (V) mM/min (x10-3)

1/[S] mM-1

1/[V] mM-1

Table 4: Tabular column for the Enzyme activity data without Inhibitor

Amount of Sucrose (ml)

Amount of Phosphate buffer (ml)

Amount of Enzyme (l)

OD at 540nm

Table 5: Tabular column for the Line-weaver Burke graph for reaction without inhibitor

[S] mM

[P] mM

Tine (min)

Velocity (V) mM/min (x10-3)

1/[S] mM-1

1/[V] mM-1

Graphs to be plotted: 1. Lineweaver- Burk plot (1/[S] vs 1/[V]). 2. Vmax vs. [I].

Result:

Exp. No: Date: STUDY OF DEACTIVATION KINETICS OF ENZYME INVERTASE Aim: To study the deactivation kinetics of Invertase enzyme. Theory:The rate of enzyme catalyzed reaction increases with temperature upto a certain limit. Above a certain temperature, the enzyme activity decreases with temperature due to enzyme denaturation. The kinetics of Thermal denaturation can be expressed as,
Where Kd is the deactivation constant. Kd varies with temperature according to the Arrhenius equation,

Where Ed is the deactivation energy and varies between 40-130 kcal/gmol Procedure:

200 l of Invertase in 4 test tubes/eppendorfs was heated and maintained at 65C for 5, 10, 15 & 20 minutes. It was then brought to room temperature and 3ml of 50mM sucrose solution was added to it and reaction was allowed to proceed for 20 minutes. In a separate test tube used as control, invertase not subjected to heat treatment was allowed to react with sucrose for 20 minutes at room temperature. Reactions in all the test tubes are stopped by boiling in a water bath for 5 minutes.

Glucose concentration was analyzed using GOD-POD kit. Observation: Table 1: Glucose Volume l Quantity g Absorbance OD at 540 nm

Table 2: Details of heating time (deactivation kinetics) Sl. No. Amount of Invertase Time at 65oC (min) Amount of Sucrose (ml) Absorbance at 540nm [P] mM Velocity of reaction (mM/min)

Vmax = Kcat*E0 at time 0 min and Vmax = Kcat*Eact at time 5, 10, 15, and 20 minutes.

Table 3: Activation Energy values Eactivation lnE

Graphs to be plotted: 1. Plot Eactive vs. time and calculate kd.

Inference and Result

Exp. No: Date:

Kinetics of enzyme Immobilization

Aim: To study the effect of immobilization of Invertase in calcium alginate beads and compare with the unimmobilized enzyme kinetics. Theory:
An immobilized enzyme is an enzyme that is attached to an inert, insoluble material such as calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme solution with calcium chloride). This can provide increased resistance to changes in conditions such as pH or temperature. It also allows enzymes to be held in place throughout the reaction, following which they are easily separated from the products and may be used again. It is recognized that the kinetic constants measured with immobilized enzymes are not true kinetic constants equivalent to those obtained in homogeneous reactions. They are apparent values because of the effects of diffusion and partitioning. There is usually a decrease in specific activity of an enzyme upon immobilization, and this can be attributed to denaturation of the enzymatic protein caused by the coupling process. Once an enzyme has been immobilized, however, it finds itself in a micro environment that may be drastically different from that existing in free solution.

Materials Required: Sucrose: 50mM Invertase: Invertase prepared by sonication of 1% Bakers yeast in Phosphate buffer pH 7 and is the stock solution. 1 ml of stock solution is diluted to 100 ml of buffer which is the invertase solution Sodium alginate: 1g in 100ml of invertase solution 6% (w/v) CaCl2 solution is prepared.

Procedure: Preparation of Immobilized enzyme: This viscous solution of 1% sodium alginate in Invertase solution was made to fall in drops into a 6% CaCl2 solution using a peristaltic pump or syringe, to form the beads. A known number of beads were taken and reaction was carried out in 50ml sucrose solution. Beads were incubated for 20 minutes. Glucose concentration was estimated using GOD/POD kit.

Determination of void volume and bead size: Beads were taken up to a certain height in the measuring jar. All voids were emptied i.e. the CaCl2 solution was decanted off. Water was then added to the same height. Water was decanted into another measuring jar and this volume is the void volume, Vv. Volume occupied by beads = Vtot Vv The number if beads was counted = N Volume of each bead Vb = 4/3 rb The radius of the bead is thus determined.

Unimmobilized system. From the number of beads taken for the experiment and the calculated value of radius of bead, the total amount of enzyme in the immobilized system was known. The same amount of enzyme was taken from the lysed yeast cells and free enzyme kinetics under identical conditions (as for immobilized kinetics) was carried out.

Observation: Table1: Immobilized system No of beads N Amount of Invertase in the beads (g) OD at 20min Concentration of Glucose g/l/M (x10-3) V (amount of glucose formed) g/l min mM /min

Table2: Unimmobilized system. Amount of enzyme in the immobilized beads ml g OD at 20min Concentration of Glucose g/l/M (x10-3) Velocity g/l /min mM/min (x10-4) (x10-2)

Result: The velocities calculated are as follows: Vimmobilized = Vunimmobilized =

Exp. No: Date: STUDY OF GROWTH KINETICS IN SHAKE FLASK CULTURE

Aim: To study the growth pattern of E. coli in batch culture using shake flask. To determine the maximum specific growth rate during log phase and apparent biomass yield coefficient.

Theory :
The growth curve of an E. coli culture can be divided into distinct phasesLag phase occurs after dilution of the starter culture into fresh medium.Cell division is slow as the bacteria adapt to the fresh medium. After 45 hoursthe culture enters logarithmic (log) phase, where bacteria grow exponentially.Cells enter stationary phase (~16 hours) when the available nutrients are used up.The cell density remains constant in this phase. Eventually the culture enters the Phase of decline, where cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded

Materials required Strain Used: E. coli DH5 Medium Used: glucose yeast extract NH4Cl K2HPO4 KH2PO4 NaCl 1M MgSO4.7H2O trace elements Trace elements composition: FeSO4 Al2(SO4)3 CuSO4 H3BO3 MnCl2 NiCl2 Na2MoO4 ZnSO4 50 mg/l 10mg/l 2mg/l 1mg/l 20mg/l 1mg/l 50mg/l 5mg/l

5 g/l 5 g/l 1 g/l 5 g/l 3 g/l 0.5 g/l 0.5 g/l 1 ml/l

Medium is prepared by autoclaving the following medium components separately and added to 250 ml shake flask to make 50 ml of medium under sterile conditions in the Laminar flow hood. 1. glucose 2. yeast extract with other salts mentioned above 3. magnesium sulphate 4. Trace metals Paramaters to be measured: a. OD at 600nm. b. Dry weight using pre-weighed eppendorf tubes b. Residual glucose analysis of the supernatant Procedure: 50 ml of medium was prepared in 250ml shake flask. 500l of inoculum from an overnight culture grown in Luria Bertani medium was added to the shake flask. Incubation was done in shaker at 37C and 200 rpm. Every hour, 1ml of culture was taken for measuring O.D at 600 nm with medium as blank. Another 1 ml of culture was withdrawn from the shake flask and centrifuged at 10,000 rpm for 5min at 4C. The pellet and supernatant were stored at -20C in the freezer for further analysis. For dry weight analysis, the pellets of last three hour sample were taken for analysis. The pellet stored in pre-weighed eppendorf tubes were washed by re-suspending in 1 ml of 0.8% saline and centrifuged at 10,000 rpm for 5min at 4C. The pellet was dried at 90C in oven for 20-24 hours. After removing from the oven, the eppendorf tubes containing the dried pellet was cooled in a dessicator and weighed. Residual glucose analysis was done by using the GOD-POD kit .The test can be performed either using cuvette in a spectrophotometer or in a 96 well plate reader. For measuring glucose standard, 2 to 10 g of glucose for 200 l of reaction mixture was taken using stock solution of 1mg/ml. And 10 to 100 g of glucose for 1000 l of reaction mixture. Assay was done as per protocol given in the kit. The sample is incubated at room temperature for 30 min. Absorbance were measured at 492 nm against buffer prepared using the kit as blank. Values were computed using standard graph.

Formulae used 1. = ln OD2 ln OD1 t2 t1

Where = specific growth rate (1/h) t = time at which the OD was measured in (h) 2. Y X/S = X2 X1 S2 S1 Where Y X/S = measured biomass yield coefficient with respect to glucose X = biomass in (g/l) S = residual glucose concentration in (g/l)

Table 1.0 Glucose Volume l Quantity g Absorbance OD at 540 nm

Table 2.0
Sl. No. Time (hour) OD (600nm) ln OD (specific growth rate) 1/hr

Graphs to be plotted OD vs time ln OD vs time OD vs dry weight OD at 492nm vs standard glucose (g of glucose ) Inferences and Result

Exp. No: Date: PLACKETT BURMAN DESIGN OF MEDIA OPTIMISATION Aim: To determine the combination of independent variables at appropriate levels so as to achieve maximum specific growth rate. Theory:
Plackett Burman is the design for optimising multifactorial experiments. It is used in screening multicomponents at two levels. It will give the range at which you have to optimize the experiment further. But this design will not give optimum concentration of the variable. It is useful for more than five variables. Here n no. of experiments will be conducted for n-1 variables. Where n is the multiples of 4 like 8,12,16,20,.100. Dummy variables are used in this process to estimate the experimental error and they have no influence in the process. Minimum one or two dummy variables should be included in the experimental set. More can be included if the real variables are less.

Materials and Methods Strain used: E. coli DH5 Medium used

Sl.No Medium High g/l 1 2 3 4 5 6 7 glucose yeast extract Cas aminoacid Ammonium Chloride Sodium Phosphate Glycine Magnesium Sulphate 4 1 0.5 2 3 1 0.5 Low g/l 0.5 0 0 0.5 3 0 0.5

Stock Concentration of Media components. Medium Concentration (g/l) Glucose 200 Yeast extract 100 Cas amino acid 50 NH4Cl 100 Glycine 100 MgSO4.7H2O 100 Prepare all the stock solution in 3g/l of phosphate buffer.

Quantity of stock solution to be added to make up 20 ml of medium. Trial glucose l 400 50 50 400 50 400 400 50 Yeast ext l 200 200 0 0 200 0 200 0 Casa. acid l 200 200 200 0 0 200 0 0 Amm.chl. phos. l 100 400 400 400 100 100 400 100 Glycine l 0 200 0 200 200 200 0 0 Mag.sul. l 50 50 50 50 50 50 50 50 Vol. * l 950 1100 700 1050 600 950 1050 200

1 2 3 4 5 6 7 8

Vol* is the volume of phosphate buffer to be removed from the shake flask before addition of stock solution of media components. Table 3:

Trial 1 2 3 4 5 6 7 8

glucose Yeast H L L H L H H L H H L L H L H L

Casa. acid H H H L L H L L

Amm.chl. L H H H L L H L

phos. H L H H H L L L

Glycine Mag.sul. L H L H H H L L H L H L H H L L

Vol

Procedure: 20 ml of medium was prepared in 100ml shake flask Medium is prepared by autoclaving 3 g/l of sodium phosphate buffer at pH of 7.0 and the remaining components of the medium were sterilized separately and added to make up 20 ml of medium in 100 ml shake flask. 1000l of inoculum from an overnight culture grown in Luria Bertani medium was added to the shake flask. Incubation was done in shaker at 37C and 200 rpm.

Every hour, 1ml of culture was taken for measuring O.D at 600 nm with medium as blank.

Observation: Table 4:

Glu A(H) A(L) Effect Mean Sq. Exp. Error F-test Table 5:
S.No Time

Yeast

CAS

NH4Cl.

Phos

Gly

MgSO4

OD

LnOD

1/hr

Formulae used = ln OD2 ln OD1 t2 t1 Where = specific growth rate (1/h) t = time at which the OD was measured in (h)

Effect on each component: [ A(H) - A (L)] 4 Mean square: [ A(H) - A (L)]2 8 Experimental error = [mean square of phosphate + mean square of MgSO4 ] 2 F- test = Mean square Experimental error

Graphs to be plotted OD vs time ln OD vs time Inference and Result