EXPT. NO.

12 ISOLATION AND ASSAY OF AMYLASE ENZYME FROM GERMINATING SEEDS INTRODUCTION Enzymes are protein catalysts necessary for cellular functions in all biological systems. They are highly specific for their substrates and function in aqueous media under very mild conditions of temperature and pH without itself undergoing changes. Amylases are a family of enzymes that degrade starch into smaller disaccharides and glucose under hydration. Maltose and glucose (reducing sugars) liberated can be estimated by means of 3,5-Dinitro Salicylic Acid (DNS). -amylase catalyses the hydrolysis of ,1 4 links of starch with the production of reducing sugars. The alkaline solution of 3, 5 DNS is reduced to 3-amino, 5-nitro salicylic acid. Amylase is found in the saliva and in the digestive tract of animals. Plant seeds often contain a reserve of rather dry insoluble starch and when a seed germinates, it produces amylase enzyme in order to convert the starch into soluble sugars which gets transported to the growing tips of roots and shoots of the developing plant. Enzyme assay Laboratory method to measure enzymatic activity, vital for study of enzyme kinetics and inhibition. Enzyme Kinetics the study of the rate at which an enzyme works. It is the determination of the rate of a reaction and how it responds to changes in experimental parameters such as enzyme concentration, substrate concentration, temperature, pH etc.

AIM Isolation of enzyme to find out its Total Activity. Preparation of standard graph for glucose. Isolation of amylase enzyme from the source- Germinating green gram seeds. Effect of different concentrations of enzyme on enzyme activity. Effect of different temperatures on enzyme activity. MATERIALS REQUIRED Test tubes Pipettes Beakers Thermometer Germinating green gram seeds Mortar and Pestle Ice Pail Cheese Cloth Centrifuge Centrifuge Tubes Water Bath Colorimeter Cuvettes Graph Paper (3 Nos) Calculator

REAGENTS 0.1M Phosphate buffer pH 6 Na2HPO4- 1.42gm in 100ml Distilled water (A) NaH2PO4 1.38gm in 100ml Distilled water (B) 69ml A+ 31ml B = 100ml Phosphate buffer Substrate Starch = 1gm in 100ml Distilled water (A) Acetic acid = 0.6ml in 100ml Distilled water (B) Mix A & B and adjust pH 4.7. (Acetate buffer). DNS (Dinitro salicilate) Solution I = 0.5gm DNS in 10ml 2M NaOH (2M NaOH = 2gm NaOH in 25ml Distilled water) Solution II = 15gm sodium potassium tartarate in 25ml Distilled water. Dissolve solution I & II separately with warming and mix I & II make up to 50ml using DW. Stock- Standard Solution 100mg of Glucose is weighed and made up to 100ml using distilled water. Working Standard Solution 50ml of the stock solution of standard is pipetted out and made up to 100ml using distilled water.

PROCEDURE I. SAMPLE PREPARATION Take 1gm germinated green gram seeds. Homogenize in 0.1 M Phosphate buffer using pre cold mortar and pestle Filter using a cheese cloth. Centrifuge at 1200rpm for 15 minutes. Supernatant was collected, kept in cold condition (in ice) and used as test solution.

II.

PROCEDURE FOR ESTIMATION OF TOTAL ACTIVITY

For the preparation of standard graph, pipette out 0.2, 0.4, 0.6, 0.8 and 1ml of working standard (Glucose) into a series of test tubes labeled S1- S5. Make up the volume in each test tube to 2ml using distilled water. 2ml distilled water will serve as blank . Pipette out 1 ml of the test solution (Extracted enzyme) into a test tube labeled as T. Add 1ml of substrate (acetate buffer) to the test solution and incubate at room temperature for 20 minutes. Terminate the reaction by adding 2ml DNS to standard, blank and test solution and heated for 5minutes in a boiling water bath. Cooled and made upto 10ml using distilled water. Read the OD at 560nm and present it in the form of a table and plot a Standard graph. Explain the observation. OBSERVATION (Write the table and calculation on the left hand side of the record)
Incubate at room temperature for 20 minutes Vol. of working std (mL) Conc. of working std (mg) Vol. of D.W. (mL) Vol. of substrate (mL) Heated for 5 minutes in a boiling water bath. Made up to 10mL using D.W. Vol. of extracted enzyme (mL) Vol. of DNS (mL) O.D . at 560 nm

Sl.No.

B S1 S2 S3 S4 S5 T

0 0.2 0.4 0.6 0.8 1

-

0 0.1 0.2 0.3 0.4 0.5

-

2 1.8 1.6 1.4 1.2 1 0

2 2 2 2 2 2 2

CALCULATION Total activity = Conc. of std. X OD of sample OD of std. RESULTS (write on the right hand side of the record) Total activity of the given enzyme sample = units/gram tissue = units/gram tissue

Volume of sample

III.

EFFECT OF TEMPERATURE ON ENZYME ACTIVITY

Take 1ml each of test solution (extracted enzyme) in 5 test tubes labeled T1T5 Add 1ml of substrate to the above test tubes Keep T1 in 0oC Keep T2 in 40C Keep T3 in 270C Keep T4 in at 500C Keep T5 at 1000C Incubate for 20 minutes Add 2ml DNS 1ml Distilled water, 1ml substrate and 2ml DNS will serve as Blank. Heat in boiling water bath for 5 minutes. Cool it and make up to 10ml with distilled water Read the OD at 560nm Plot a graph, and explain the observation with the help of a table.

OBSERVATION (Write the table on the left hand side of the record)
Vol. of extracted enzyme (mL) Vol. of substrate (mL) Vol. of D.W. Vol. of DNS (mL) Heated for 5 minutes in a boiling water bath. Made up to 10mL using D.W. for 20 minutes at Incubate different temperatures O.D. at 560 nm

Sl.No.

B T1(0oC) T2(40C) T3(270C) T4(500C) T5(1000C) RESULTS

0 1 1 1 1 1

1 1 1 1 1 1

1 0 0 0 0 0

2 2 2 2 2 2

( write on the right hand side of the record)

IV.

EFFECT OF DIFFERENT CONCENTRATIONS OF ENZYME ON ENZYME ACTIVITY

Take supernatant (test solution) in 5 test tubes C1 C5. 0.5, 1, 1.5, 2, and 2.5ml respectively & make up to 2.5ml using distilled water. Add 1ml of substrate and incubate for 20 minutes. Terminate the reaction by adding 2ml DNS to test and blank 2.5 ml distilled water,1ml substrate and 2ml DNS will serve as Blank Heat for 5 minutes in boiling water. Cool and make up to 10ml using distilled water. Read the OD at 560 nm. Plot a graph and explain the observation with the help of a table.

OBSERVATION (Write the table on the left hand side of the record)

Sl.No.

Vol. of extracted enzyme (mL)

Vol. of D.W.

Vol. of substrate (mL)

Vol. of DNS (mL)

Heated for 5 minutes in a boiling water bath. Made up to 10mL using D.W.

for 20 minutes at Incubate different temperatures

O.D. at 560 nm

B C1 C2 C3 C4 C5 RESULTS

0 0.5 1 1.5 2 2.5

2.5 2 1.5 1 0.5 0

1 1 1 1 1 1

2 2 2 2 2 2

(write on the right hand side of the record)