Bio Chem Lab Report 01

7/23/2019 Bio Chem Lab Report 01

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BIOC 12141

PREPARATION OF CRUDE INVERTASE

EXTRACT

&

DETERMINATION OF SPECIFIC ACTIVITY

OF INVERTASE

Student Name : I.W. Chathuranga Chandrasiri

Admission Number : BS/2012/181

Perform Date : 15/08/2012

Submission Date : 22/08/2012


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Date : 2014.08.15

Experiment No : 01

Experiment title :

Preparation of crude invertase extract and

Determination of specific activity of invertase

Objectives :

To achieve the ability to prepare an enzyme extract.

To understand the enzyme units and specific activity.

Introduction :

Proteins form the class of biological macromolecules that have the most well-defined

physicochemical properties, and consequently they were generally easier to isolate and

characterize than nucleic acids, polysaccharides, or lipids. Furthermore, proteins, particularly

in the form of enzymes, have obvious biochemical functions. The central role that proteins

play in biological processes has therefore been recognized since the earliest days of

biochemistry.

Theory :

Protein extraction

Cellular location of the protein to be purified determines the procedures that can beused to obtain an extract containing the protein in soluble form. Extracellular proteins such

as those fermentation broths or in serum simple removed from insoluble material from

centrifugation or filtration. Intracellular enzymes and those found in the organelles of

eukaryotes may be released into extract by general cell disruption techniques.

Membrane bound proteins present a greater problem for isolation because they

normally cannot be released by simple disruption procedures. Protein associated with

phospholipids and protein liquid complex must be dissociated before the extraction. Extrinsic

membrane protein those bound only to the surface membrane can generally release byraising ionic concentration 1M NaCl or by freezing and thawing. Intrinsic membrane protein

those embedded in the membrane best released by treatment with either a detergent such

as SDS or organic solvents such as butanol. In the all cases precaution must be taken to

minimize protein denaturation and proteolysis.

Protein Purification

To be able to isolate a specific protein from a crude mixture the physical and/orchemical properties of the individual protein must be utilized. There is no single or simple

way to purify all kinds of proteins. Procedures and conditions used in the purification process


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of one protein may result in the inactivation of another. The final goal also has to be

considered when choosing purification method.

The purity required depends on the purpose for which the protein is needed. For an

enzyme that is to be used in a washing powder, a relatively impure sample is sufficient,

provided it does not contain any inhibiting activities. However, if the protein is aimed for

therapeutic use it must be extremely pure and purification must then be done in several

subsequent steps.

The aim of a purification process is not only removal of unwanted contaminants, but

also the concentration of the desired protein and the transfer to an environment where it is

stable and in a form ready for the intended application.

Yeast cell walls are composed of two layers of -glucan, the inner layer being insoluble

to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the

carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of

extracellular barrier confer shape and rigidity to the cells.

Plant cell walls are particularly strong, making them very difficult to disrupt

mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical

disruption using glass beads or sand, whereas bacterial cell walls are the easiest to break

compared to these other cell types.

Historically, physical lysis was the method of choice for cell disruption and extraction

of cellular contents; however, it often requires expensive, cumbersome equipment and

involves protocols that can be difficult to repeat due to variability in the apparatus (such as

loose-fitting compared with tight-fitting homogenization pestles). Also, traditional physical

disruption methods are not conducive for high throughput and smaller volumes typical of

modern laboratory research.

Enzyme units

The enzyme activity is frequently expressed in terms of units such that one unit is

amount of enzyme that catalysis the conversion of 1 micromole of substrate per minute

under defined conditions.

The SI unit of enzyme activity is katal which represents the transformation of 1 mole of

substrate per second.

1U = 1 mol min-1 1 katal = 1 mol s-1

1U = kat/60 = 16.67 nkat


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Specific activity

Purity of an enzyme is expressed in terms of the specific activity, which is the number of

enzyme units per milligram of protein.

The more purified an enzyme preparation is the greater is its specific activity.

Specific activity =number of enzyme units (U)

Number of mg of total protein=

U

Materials :

Chemicals

o Glucose solution

o H2O

o

Nelsons reagento Arsenomolybdate reagent

o Bakers yeast

o pH 5 citrate buffer

o Sucrose solution (1%)

o BSA solution

Procedure :

Extraction of invertase

1g of bakers yeast was taken into a motor and a little amount of sand added in to

it.Citrate buffer (pH 5, 100 mM, 2.0 mL) was also added. The mixture was grinded very well

until become slurry. Then buffer (3.0 mL) was added and mixed well. The mixture was

centrifuged at 5000g for 15 min at 40 C.

Invertase assay

Sucrose (1%,0.1 mL ) and invertase crude solution(100uL)was taken and citrate buffer

(100Mm,pH 5) required amount was added, to make the final volume of the tube 2.00

mL.The mixture was incubated for 5 min at 370 C. Nelsons procedure was followed to

determine the amount of reducing sugars.

Nelsons procedure

Nelsons reagent (1.0 mL) was added to stop the reaction. The tube was boiled for 20

min. Cooled to room temperature and arsenomolybdate reagent (1.0 mL) was added. Tubes

were mixed well and allowed to stand for 5 min.Distilled water (7.0 mL) was added and mixed

and the absorbance was measured. Blank experiment was also carried out without adding

the enzyme.

Glassware

o Test tubes.

o Mortar

o

Micro pipettes.o

Droppers

o Spectrophotometer


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Tube No

1 2 3 4 5 6 7 8 9 10

Glucose(4mM)- 0.02 0.05 0.1 0.15 0.2 0.25 0.3 - -

Fructose(4mM)- - - - - - - - 0.2 -

Sucrose(4mM)- - - - - - - - - 0.2

H2O1.0 0.98 0.95 0.90 0.85 0.8 0.75 0.7 0.8 0.8

Nelsons reagent1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Cover the tubes with cotton wool. Boil for 20 min. cool tubes to room temperature

Arsenomolybdate

reagent

1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Mix tubes on a Vortex mixer and allow to stand for 5 min

Water7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0

Mix on votex mixture

Dilution


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Slandered curve for the determination of proteins.

Addition 1 2 3 4 5 6

Albumin bourn serum (1 mg/mL) 1.0 2.0 3.0 4.0 5.0 -

Enzyme crude solution - - - - - 1.0

Biuret reagent 2.0 2.0 2.0 2.0 2.0 2.0

H2O 4.0 3.0 2.0 1.0 - 4.0

Result :

Absorbance of the invertase assay

Absorbance for the Blank = 0.90

Absorbance for the sample = 0.83

Absorbance of the reducing sugars

Sample Absorbance

1 0.07

2 0.20

3 0.32

4 0.58

5 0.67

6 0.79

7 0.82

8 0.85

9 0.63

10 0.64


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Absobance of the yeast invertase test

sample Absorbance

1 0.17

2 0.20

3 0.23

4 0.28

5 0.32

6 0.24

Calculation:

2 = 4.0 10

0.02 10

10 10

= 8 x 10-6 mol L-1

3 =4.0 10 0.05 10

10 10

= 20 x 10-6 mol L-1

4 = 4.0 10

0.10 10

10 10

= 40 x 10-6 mol L-1

5 =4.0 10 0.15 10

10 10

= 60 x 10-6 mol L-1

6 =

4.0 10 0.20 10

10 10

= 80 x 10-6 mol L-1

7 =4.0 10 0.25 10

10 10

= 100 x 10-6 mol L-1

8 =

4.0 10 0.30 10

10 10 = 120 x 10-6 mol L-1


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Standard curve for determination of reducing sugars

Tube number Concentration mol/L absorbance

1 0.07

2 8.0 x 10-6 0.20

3 20.0 x 10-6 0.32

4 40.0 x 10-6 0.58

5 60.0 x 10-6 0.67

6 80.0 x 10-6 0.79

7 100.0 x 10-6 0.82

8 120.0 x 10-6 0.85

1 =1.0 1.0

7.0

= 1.42 x 10-1 mg mL-1

2 =1.0 2.0

7.0

= 2.84 x 10-1 mg mL-1

3 =1.0 1.0

7.0

= 4.28 x 10-1 mg mL-1

4 =1.0 1.0

7.0

= 5.17 x 10-1 mg mL-1

5 =1.0 1.0

7.0

= 7.14 x 10-1 mg mL-1

Standard curve for concentration of total protein

Tube number Concentration mg/mL absorbance

1 1.42 x 10-1 0.17

2 2.85 x 10-1 0.20

3 4.28 x 10-1 0.23

4 5.17 x 10-1 0.28

5 7.14 x 10-1 0.326 ? 0.24


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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

8.0 x 10-6 20.0 x 10-6 40.0 x 10-6 60.0 x 10-6 80.0 x 10-6 100.0 x 10-6 120.0 x 10-6

Absoption

Concentration (mol/L)

graph of Concentration of Glucose vs Absorption at 540nm


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0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

1.42 x 10-1 2.85 x 10-1 4.28 x 10-1 5.17 x 10-1 7.14 x 10-1

Absorbance

Concentration (mg/mL)

Graph of Concentration of Invertase vs absorbance at 540nm


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Absorption of the sample = 0.24

Concentration of protein = 4.5 x 10-1mg/mL

Total protein content = CV

= 4.5 x 10-1mg/mL x 5.0 mL

= 2.25 mg

Enzyme activity of invertase =

= 1 x 10-4U

Specific activity =number of enzyme units (U)

Number of mg of total protein=

U

Specific activity =1 10U

2.25 mg

Specific activity = 4.445 10

Discussion :

This experiment was done to determine the enzyme activity of invertase enzyme. Forthe extraction of invertase enzyme, backers yeast was taken because backers yeast contains

the invertase enzyme in its cytoplasm.

For the extraction, the yeast sample was ground with a little amount of sand to break

the cell walls of the yeast for the whole amount of enzyme to be taken out.

A buffer was added to keep the mixture in a constant pH value because the enzymes

are denatured due to changes of the pH value and so that the enzyme activity of invertase

cannot be determined.

Then the mixture was centrifuged to separate the crude invertase enzyme form the

cell debris. The extracted enzyme is called a crude enzyme because the supernatant contains

not only the pure enzyme but also the other cell components as well.

Nelson method is an indirect colorimetric procedure that can be used to determine

the concentration of reducing sugars. Standard series of D-glucose series is used to establish

a standard curve and the amount of reducing sugars present in unknown solution is

determined from the standard curve. When glucose is heated in an alkaline solution


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containing Cu2+(Nelson reagent) glucose is oxidized and cupric ions reduced quantitatively

to cuprous ions. The tubes are covered during heating to prevent evaporation.

The blue color appears after adding arsenomolybdate is caused by the reduction of

hexavalent molybdenum atom in H3AsO4(MoO4)12 to the pentavalent state by the Cu+ ion.

This reaction is quantitative, therefore the intensity of the blue color formed is directly

proportional to the reducing sugar concentration.

The metals present in Nelson reagent and arsenomolybdate can inactive enzymes.

This property is used in the procedure to break enzymes activity. This is important since if

the enzymes activity doesnt stop it keeps hydrolyzing sucrose. Therefore cannot get a

correct value of (glucose) after a given time because the colorimeter doesnt show a proper

value.

Purity of an enzyme is expressed in terms of the specific activity, which is the number

of enzyme units per milligram of protein.

In order to measure the amount of total amount of protein present in the extracted

enzyme sample, a known amount of enzyme was reacted with biuret reagent and the

absorbance of the formed purple coloured solution was measured.

In order to calibrate the standard curve a series of standard Albumin Bovine serum was

made and the series was reacted with biuret and the absorbance of the purple coloured

series was measured using a colorimeter. With the use of the standard graph the total

amount of protein in the extracted sample can be calculated.

So that the specificity of invertase enzyme can be calculated.

Conclusion :

Specific activity of the extacted yeast invertase 4.445 10

References :

Stryer,L., Biochemistry, 4thedition,W.H. Freeman company, New York, 1999

Vote,D., Vote,J.G., Biochemistry, 4thedition, john wiley and sons inc, USA, 2011

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Bio Chem Lab Report 01