Starch Hyrolysis Of Amylase Enzyme Experiment Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Monosaccharides are most basic units of carbonhydrates.They are the simplest form of the sugar. Glucose,galactose,fructose and ribose are example for the monosaccharides. Disaccharide is form when two monosaccharides combined. Lactose and sucrose are example for the disaccharides.

Plants store glucose as the polysaccharide starch. Starch can be separated into two fractions--amylose and amylopectin. Natural starches are mixtures of amylose (10-20%) and amylopectin (80-90%). Amylose is a linear compound which is soluble in water . Aslo linked by α ( 1,4 ) glicosidic bond. Amylopectin is branched compound which is not soluble in water. Also linked by α ( 1,6 ) glicosidic bond. (1)

Figure 1 : STARCH

Figure 2 : Starch-iodine complex

Amylase is an enzyme which present in human salvia. It breaks starch down into sugar. All

amylases are glycoside hydrolases and act on α-1,4-glycosidic bonds. It will start to denature at

around 60C.

Spectrophotometer measures the transmission or absorption of liquids or solids as a function of

wavelength. Spectrophotometer is used for 2 different purpose :

To determine the absorption spectrum of a pure substance in solution

To determine the concentration of a solution

Figure 3 : Single beam spectrophotometer

% T = ( I / I O ) . 100 ABS = log 10 ( 100/ % T)

APPARATUS :

Equipments :

Test tubes

Pipettes

Pasteur pipettes

Beaker 250 mL

Test tube rack

Plastic cuvettes

Spectrophotometer

Heater

Weighing dish

Weight

Chemicals :

Human salivary enzyme

Starch solution 20 g/L

HCl stopping solution, 0.1N HCl

Iodine reagent stock solution(in aqueous solution)

Iodine : 5 g/l

KI : 50 g/l

Dilute to 1:100

Potassium phosphate buffers

KH2PO4

KH2PO4.3H2O

PROCEDURE :

Preparation of 20 g/l starch solution :

20g of soluble potato starch was mixed in approx. 50 mL of cold water.

The slurry was added to aprox. 900 mL of gently boiling water in a large beaker while stirring.

The gelatinized starch solution was mixed well and cooled to room temperature.

More water was added to bring the total volume to 1 liter.

Few drops of the starch solution was put on a glass plate. 1 drop of the iodine reagent was added and the deep blue color was seen.

Preparation of Enzyme solution :

1 mL of salvia was diluted with 9 mL water. 60 mL of 0.5 % NaCl solution was added .

Effect of the pH :

0.1 M pH buffer solutions was prepared ranging from pH=4.5 to pH=9 in increments of one pH unit.

An equal volume of one of the above buffer solutions were added to 5.0mL of the 20 g/l starch solution prepared in step 1 .The resulting solution was contained 10g/l of starch in a buffered environment.

The enzymatic digestion process was started by adding 1 mL of human salivary enzyme solution; shaked and mixed.

The hydrolysis reaction was proceeded for exactly 10 minutes at 25oC.

0.5 mL of the reacted starch solution was added to 5 mL of the HCl stopping solution.(0.1 N)

0.5 mL of the above mixture was added to 5 mL iodine solution to develop color.Shaked and mixed.

The absorbance was measured with a spectrophotometer at 620 nm.Buffer was used as a blank.

Effect of Temperature :

The temperatures of the temporary water baths in 250 mL beakers was prepared and adjusted the temperatures ranging from 30oC to 90oC in increments for 20oC.

The starch substrate was prepared by diluting the 20g/l starch solution prepared in step 1 with an equal volume of pH=7.0 phosphate buffer solution.This results in a working starch concentration of 10 g/l. 5 mL of the starch solution was added to each of test tubes.

The temperature of each of the starch solutions was allowed to come to equilibrium with that of the water bath.

1 mL of human salivary enzyme solution was added to each of the thermostated test tubes to start the reaction.The raction was stopped after exactly 10 minutes and the starch content was analizied by following the procedures outlined in step 3.

CALCULATIONS AND OBSERVATIONS :

Data tables:

Table 5.1: Effect of pH:

pH

5

6

7

8

9

Absorbance

0.038

0.054

0.049

0.022

0.080

Table 5.2: Effect of temperature:

Temperature

30

50

70

90

Absorbance

0.024

0.006

0.039

0.115

Absorbance / temperature

Absorbance / pH

DISCUSSION :

In this experiment the purpose was to hydrolyze starch with amylase enzyme and observe the effect of pH and temperature on this reaction.First we prepared the starch solution,added 1 drop of the iodine reagent and saw that a deep blu color was developed. After that we prepared the enzyme solution which salvia was used.Then we looked to the effect of pH and effect of temperature. In effect of temperature buffer solutions were prepared which ranging from pH 4.5 to pH 9. We used two different solutions because of the diffence buffering capacity of these two solutions. We used HCl and NaOH for pH. We used HCl to decrease pH and we used NaOH to increase pH also, we detected the pH by using pH meter. Each buffer with different pH values were mixed with starch solution and then salivary solution was added. Then we added iodine solution to detect whether reaction took place or not. Since if enzyme functions starch in the solution will be hydrolyzed and this will lead to have light color of the solution; absorbance will be low. Since our body is in neutral pH we expect to have light colored solution at pH 7 and dark color at pH 5, 8 and 9. The enzyme won't work in higher temperature values that can denature it like 90, 70 and maybe 50. Protein's absorbance values are expected to increase as the protein denaturizes. This can be explained by the surface of reflection of the light is increased. Denaturized form of protein has higher possibility to be interacted with the light from the spectrophotometer and thus absorbance will increase.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.