RATIFICATION PAGE

Complete report of Basic Biology with the title “Influence of PH to Enzyme

Activities”, created by :

name : Bertha Tandi

reg. Number : 1414442010

group : V

class : ICP B Biology

after it’s checked and consulted by Assistant and Assistant Coordinator, it has

fulfilled requirement.

Makassar, January 2014

Assistant Coordinator Assistant

Djumarirmanto S.Pd Muh. Nur ArsyadID . 09104158

Known by,Lecturer of Responsibility

Drs. H. Hamka L.MsNIP. 19621231 198702 1 005

CHAPTER IINTRODUCTION

A. Background

Metabolism includes all the chemical reactions that occur in the body of

organisms. The metabolic process is the enzymatic reaction, meaning that the

reaction involving the enzyme. Enzymes are substances in each of the reactions in

living organisms that act as biocatalisator, which is a catalyst in chemical

reactions of living cells. As a catalyst, the enzyme is a chemical agent that can

change the rate of a reaction without he himself had to change because of the

reaction. With the enzyme, the traffic through the chemical - metabolic pathways

run more smoothly. In these reactions are described substances and substances

that are formed, the substances described by the reaction are called substrates, and

the newly formed from the reaction are called products. 

In a chemical reaction, the enzyme did not change shape, do not affect the

balance of the reaction or change the product. The reaction of complex chemical

reactions in the body will last a very slow if without enzymes. This enzyme works

in a liquid aqueous solution, temperature, and pH are in accordance with the

biological physiological conditions. Through its activities, the enzymes are well

coordinated so as to produce a good relationship between a number of different

metabolic activity, which refers to support in our life. Enzyme not determine the

direction of the reaction (back - back), can be used repeatedly for not damaged,

needed in small amounts. The enzyme has a different temperature and pH -

different to be able to work optimally, because if the temperature and acidity are

not in accordance with the nature of an enzyme, the enzyme cannot work

optimally, inactive, even experienced denaturation (damage). The enzyme can be

found in animals and plants. One such enzyme is an enzyme amylase (diastase)

contained in the plant. Enzyme can hydrolyze starch into sugar. Amylase

produced by the leaves or seeds which are for prove the above statement, we

conducted an experiment entitled "effect of pH on enzyme activity". 

B. Purpose

The purpose of this practicum, to showing influenced pH about amylase

enzyme activity.

C. Benefit

Based on this practicum the benefit of this practicum the university

student will know about influenced pH about amylase enzyme activity.

CHAPTER IIPREVIEW OF LITERATURE

An enzyme is a macromolecule that acts as a catalyst, chemical agent that

speeds up a reaction without being consume by the reaction. (In this chapter, we are

focusing on enzymes that are proteins. RNA enzymes, also called ribozymes, without

regulation by enzymes, chemical traffic through the pathways of metabolism would

become terribly congested because many chemical reactions would take such a long

time. In the next two sections, we will see what prevents a spontaneous reaction from

occurring faster and how an enzyme changes the situation (Campbell, 2011: 152).

Enzymes can be found both in animals and in plants. One of the enzymes

found in plants is amylase. Other names of amylase is amylase hydrolyze latase. The

enzyme can amylase hydrolyze starch into sugar. Amylase produced by the leaves or

seeds germinating. Amylase activity is affected by inorganic salts, pH, temperature,

and light. The optimum pH of amylase hydrolyze according to Hopkins, Cole, and

Green is 4.5 to 4.7 (Tim Dosen, 2014: 32).

An enzyme catalyzes a reaction by lowering the EA barrier enabling the

reactant molecules to absorb enough energy to reach the transition state even at

moderate temperatures. An enzyme cannot change the ΔG for a reaction; it cannot

make an endergonic reaction exergonic. Enzymes can only hasten reactions that

would eventually occur anyway, but this function makes it possible for the cell to

have a dynamic metabolism, routing chemicals smoothly through the cell’s metabolic

pathways. And because enzymes are very specific for the reactions they catalyze,

they determine which chemical processes will be going on in the cell at any particular

time. The reactant an enzyme acts on is referred to as the enzyme’s substrate. The

enzyme binds to its substrate (or substrates, when there are two or more reactants),

forming an enzyme substrate complex. While enzyme and substrate are joined, the

catalytic action of the enzyme converts the substrate to the product (or products) of

the reaction. The overall process can be summarized as follows:

Enzyme _ Enzyme- Enzyme _

Substrate(s) Δ substrate Δ Product(s) complex

For example, the enzyme sucrase (most enzyme names end in -ase) catalyzes the

hydrolysis of the disaccharide sucrose into its two monosaccharides, glucose and

fructose.

Sucrase _ Sucrase- Sucrase _

Sucrose _ Δ sucrose-H2O Δ Glucose _H2O complex Fructose

The reaction catalyzed by each enzyme is very specific; an enzyme can recognize its

specific substrate even among closely related compounds. For instance, sucrase will

act only on sucrose and will not bind to other disaccharides, such as maltose. What

accounts for this molecular recognition? Recall that most enzymes are proteins, and

proteins are macromolecules with unique three-dimensional configurations. The

specificity of an enzyme results from its shape, which is a consequence of its amino

acid sequence (Campbell, 2011: 152).

Most enzymes are active only over a narrow pH range and have an optimal

pH, at which the rate of reaction is fastest. The optimal pH for most human enzymes

is between 6 and 8. (Recall from Chapter 2 that buffers minimize pH changes in cells

so that the pH is maintained within a narrow limit.) Pepsin, a protein digesting

enzyme secreted by cells lining the stomach, is an exception; it works only in a very

acidic medium, optimally at pH 2. In contrast, trypsin, a protein-splitting enzyme

secreted by the pancreas, functions best under the slightly basic conditions found in

the small intestine. The activity of an enzyme may be markedly changed by any

alteration in pH, which in turn alters electric charges on the enzyme. Changes in

charge affect the ionic bonds that contribute to tertiary and quaternary structure,

thereby changing the protein’s conformation and activity. Many enzymes become

inactive, and usually irreversibly denatured, when the medium is made very acidic or

very basic (Salomon, 2008: 164).

Effects of Temperature and pH Recall from Chapter 5 that the three-

dimensional structures of proteins are sensitive to their environment. As a

consequence, each enzyme works better under some conditions than under other

conditions, because these optimal conditions favor the most active shape for the

enzyme molecule. Temperature and pH are environmental factors important in the

activity of an enzyme. Up to a point, the rate of an enzymatic reaction increases with

increasing temperature, partly because substrates collide with active sites more

frequently when the molecules move rapidly. Above that temperature, however, the

speed of the enzymatic reaction drops sharply. The thermal agitation of the enzyme

molecule disrupts the hydrogen bonds, ionic bonds, and other weak interactions that

stabilize the active shape of the enzyme, and the protein molecule eventually

denatures. Each enzyme has an optimal temperature at which its reaction rate is

greatest. Without denaturing the enzyme, this temperature allows the greatest number

of molecular collisions and the fastest conversion of the reactants to product

molecules. Most human enzymes have optimal temperatures of about 35–40°C (close

to human body temperature). The thermophilic bacteria that live in hot springs

contain enzymes with optimal temperatures of 70°C or higher. Just as each enzyme

has an optimal temperature, it also has a pH at which it is most active. The optimal

pH values for most enzymes fall in the range of pH 6–8, but there are exceptions. For

example, pepsin, a digestive enzyme in the human stomach, works best at pH 2. Such

an acidic environment denatures most enzymes, but pepsin is adapted to maintain its

functional three-dimensional structure in the acidic environment of the stomach. In

contrast, trypsin, a digestive enzyme residing in the alkaline environment of the

human intestine, has an optimal pH of 8 and would be denatured in the stomach

(Campbell, 2011: 155).

In an enzyme-catalyzed reaction, the reactants are called substrates. Substrate

molecules bind to a particular site on the enzyme, called the active site, where

catalysis takes place. The specificity of an enzyme results from the exact three-

dimensional shape and structure of its active site, into which only a narrow range of

substrates can fit. Other molecules—with different shapes, different functional

groups, and different properties—cannot properly fit and bind to the active site. The

names of enzymes reflect the specificity of their functions a reaction is recovered

during the ensuing “downhill” phase of the reaction, so it is not a part of the net free

energy change and often end with the suffix “-ase.” For example, the a reaction is

recovered during the ensuing “downhill” phase of the reaction, so it is not a part of

the net free energy change (Heller, 2011: 113-114).

Almost every biological process is pH dependent; a small change in pH

produces a large change in the rate of the process. This is true not only for the many

reactions in which the H_ ion is a direct participant, but also for those in which there

is no apparent role for H_ ions. The enzymes that catalyze cellular reactions, and

many of the molecules on which they act, contain ionizable groups with characteristic

pKa values. The protonated amino and carboxyl groups of amino acids and the

phosphate groups of nucleotides, for example, function as weak acids; their ionic

state depends on the pH of the surrounding medium. As we noted above, ionic

interactions are among the forces that stabilize a protein molecule and allow an

enzyme to recognize and bind its substrate. Cells and organisms maintain a specific

and constant cytosolic pH, keeping biomolecules in their optimal ionic state, usually

near pH 7. In multicellular organisms, the pH of extracellular fluids is also tightly

regulated. Constancy of pH is achieved primarily by biological buffers: mixtures of

weak acids and their conjugate bases. We describe here the ionization equilibria that

account for buffering, and we show the quantitative relationship between the pH of a

buffered solution and the pKa of the buffer. Biological buffering is illustrated by the

phosphate and carbonate buffering systems of humans. Although many aspects of cell

structure and function are influenced by pH, it is the catalytic activity of enzymes that

is especially sensitive. Enzymes typically show maximal catalytic activity at a

characteristic pH, called the pH optimum). On either side of the optimum pH their

catalytic activity often declines sharply. Thus, a small change in pH can make a large

difference in the rate of some crucial enzyme-catalyzed reactions. Biological control

of the pH of cells and body fluids is therefore of central importance in all aspects of

metabolism and cellular activities (Salomon, 2008: 160).

PH affects enzyme activity the rates of most enzyme-catalyzed reactions

depend on the pH of the medium in which they occur. Each enzyme is most active at

a particular pH; its activity decreases as the solution is made more acidic or more

basic than its “ideal” (optimal) pH several factors contribute to this effect. One is the

ionization of carboxyl, amino, and other groups on either the substrate or the enzyme.

In neutral or basic solutions, carboxyl groups (—COOH) release H+ to become

negatively charged carboxylate groups (—COO–). Similarly, amino groups (—NH2)

accept H+ ions in neutral or acidic solutions, becoming positively charged —NH3+

groups (see Chapter 2). Thus, in a neutral solution, a molecule with an amino group is

attracted electrically to another molecule that has a carboxyl group, because both

groups are ionized and the two groups have opposite charges. If the pH changes,

however, the ionization of these groups may change. For example, at a low pH (high

H+ concentration), the excess H+ may react with the —COO– to form COOH. If this

happens, the group is no longer charged and cannot interact with other charged

groups in the protein, so the folding of the protein may be altered. If such a change

occurs at the active site of an enzyme, the enzyme may no longer have the correct

shape to bind to its substrate (Heller. 2011: 122).

Cells regulate the rates of chemical reactions with enzymes, which are

biological catalysts that increase the speed of a chemical reaction without being

consumed by the reaction. Although most enzymes are proteins, scientists have

learned that some types of RNA molecules have catalytic activity as well . The

catalytic ability of some enzymes is truly impressive. For example, hydrogen

peroxide (H2O2) breaks down extremely slowly if the reaction is un catalyzed, but a

single molecule of the enzyme catalase brings about the decomposition of 40 million

molecules of hydrogen peroxide per second! Catalase has the highest catalytic rate

known for any enzyme. It protects cells by destroying hydrogen peroxide, a

poisonous substance produced as a by-product of some cell reactions. The

bombardier beetle uses the enzyme catalase as a defense mechanis (Salomon, 2008:

163).

CHAPTER IIIOBSERVATION METHOD

A. Place and Date

Day / date : Monday/ January th 2010

Time : 16.00 Wita – 18.00 Wita

Place : Laboratory of Biology, 3rd west floor Mathematics and Science

Faculty State University of Makassar

B. Tools and Materials

1. Tools

a. 10 pieces Test tube

b. 1 piece Test tube rack

c. 5 pieces Dropping pipette

d. 1 piece Bunsen burner

e. 3 pieces Universal indicator

f. 1 piece Beaker glass

2. Material

a. Extract of phaseolus sprout

b. Amylum solution

c. Fehling solution A and B

d. HCl solution (10%)

e. NaOH solution (1%)

f. PH paper

g. Filter paper

h. Aquadest

i. 1 piece Matches

C. Work Procedure

1. Prepared 10 units of test tube and its rack. Divided the tubes in to 4 groups

(Tube A, B, C, and D). Tube A, B and C were classified in to 3 tubes and

gave them (A1/B1/C1, A2/B2/C2, And A3/B3/C3). For tube D, it was

independent.

2. For A tubes, dropped 2 drops of starch solution and then dropped 1 mL of

extract.

3. Added 2 drops of fehling A and B, measured the solution pH using pH meter.

Observed the initial color.

4. Let the 3 tubes about a few minutes. A1 during 5 minutes, A2 during 10

minutes, A3 during 15 minutes. After that heated each tubes.

5. After heating the tubes, observed each colors.

6. Wrote down your observation.

7. For B tubes, dropped 2 drops of starch solution and then dropped 1 mL of

extract, after that added 2 drops of NaOH, done the same procedures from

step 3 to step 6.

8. For C tubes, dropped 2 drops of starch HClsolution and then dropped 1 mL of

extract, after that added 2 drops of. Done the same procedures from step 3 to

step 6.

9. For D tube, dropped 2 drops o starch solution and then dropped 1 mL of

extract. Gave addition of fehling A and B about 2 drops. And then, observed

the indicial color.

10. Heated the tube, and then observed the color’s changing.

CHAPTER IVRESULT AND DISCUSSION

A. Observation Result1. Observation Table

No. Tube pH First Color Last Color

1. A

A1

12

Blue Yellow Orange

A2 Blue Orange Turbid

A3 Blue Light Yellow

2. B

B1

11

White

White Turbid +

Sediment

B2 White

White Turbid +

Sediment

B3 White

White Turbid +

Sediment

3. C

C1

1

Clear Yellow Light Yellow

C2 Clear Yellow Light Yellow

C3 Clear Yellow Light Yellow

4. D - 13 Blue Light Blue

2. Figures of Observation1) Tube I

a.Tube I A

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 2 drops of

Fehling A and B.

3. After heated for 5 minutes.1 2 3

b. Tube I B

Note :

1. The addition of 1 ml of

starch and germ extract.

2. The addition of 2 drops

of Fehling A and B.

3. After heated for 10

minutes.

1 2 3

c. Tube I C

Note :

1. The addition of 1 ml of

starch and germ extract.

2. The addition of 2 drops of

Fehling A and B.

3. After heated for 15

minutes.

1 2 3

2)Tube II

a.Tube II A

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 3 drops of

NaOH.

3. After heated for 5 minutes.

1 2 3

b.Tube II B

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 3 drops of

NaOH.

3. After heated for 10 minutes.

1 2 3

c. Tube II C

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 3 drops of

NaOH.

3. After heated for 15 minutes.1 2 3

3) Tube III

a. Tube III A

Note :

1. The addition of 1 ml of

starch and germ extract.

2. The addition of 3 drops of

HCl.

3. After heated for 5 minutes.1 2 3

b. Tube III B

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 3 drops of HCl.

3.

4. After heated for 10 minutes.1 2 3

c. Tube III C

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 2 drops of

HCl

3. After heated for 15 minutes.1 2 3

4. Tube IV

Note :

1. The addition of 1 ml of starch

and germ extract.

2. The addition of 2 drops of

Fehling A and B.

3. This direct heated.1 2 3

B. DISCUSSED

In the tube 1 in the tube given amylum, added fehling A and B, and added

green peal obtained the 12 pH and we heated the tube 1A. 1B, and 1C with

different time and the color was change in the tube 1A from blue became the

yellow orange, tube 1B from blue into orange turbir, and the 1C from dark blue

into light yellow. So for the tube 1 is basa.

In the tube 2 in the tube given amylum, added fehling A and B, added

extract green peal and 3 drop NaOH obtained the 11 pH and we heated the tube

2A. 2B, and 2C with different time and the color was change in the tube 2A from

white became the White Turbid + Sediment, tube 2B from white into White

Turbid + Sediment, and the 2C from white into White Turbid + Sediment. So it

mean the tube 2 is base.

In the tube 3 in the tube given amylum, added fehling A and B, added

extract green peal and 3 drop HCl solution obtained the 1 pH and we heated the

tube 3A. 3B, and 3C with different time and the color was change in the tube 3A

from clear yellow became the light yellow, tube 3B from clear yellow into light

yellow, and the 3C from clear yellow into light yellow. So it mean the tube 3 is

acid.

The last tube 4 wasn’t done anything just give fehling A and B. So the

result in the tube 4 is bases.

CHAPTER VCONCLUSION AND SUGGESTION

A. Conclusion

Based on the practicum about the influence the PH of enzyme activity, the

practicum draw the conclusion as follows:

The pH of the substrate it can be evidence the enzyme activity which is

enzyme function is to make fast the reaction.

B. Suggestion

In conducting the experiment, must be done seriously, meticulous when

dripping the solution until the solution is dripped do too much. Perform

experiments in accordance with workplace procedures.

BIBLIOGRAPHY

Neil A, Campbell. Reece, Jane B. 2009. Campbell biology ninth edition. Amerika

Serikat : MasteringBiology® and BioFlix®.

Orians, Heller. 2011. The Science of Biology 7Th Edition. New York: Porves Sadava.

Solomon, Eldra P. Berg, Linda R. 2008. Biology eighth edition. Amerika Serikat:

The part of Thomson Corporation.

Tim Pengajar Biologi. 2010. Penuntun Praktikum Biologi Dasar. Makassar: Laboratorium FMIPA UNM.

APPENDIX

A. Question

1. What to Fehling's solution A and Fehling B and JKJ?

2. Why the enzyme extract from the seeds need centrifuge?

3. What is the function of HCl and NaOH in this practicum?

B. Answer

1. Usefulness of Fehling's solution A and Fehling B to test whether an extract or a

solution containing glucose or not. JKJ solution is used to test whether a

solution containing starch or not.

2. Enzymes from seeds that need centrifuge mix solid substances and liquid can be

separated so that the solids can settle to the bottom while the liquid remains in

the liquid part. Or is this liquid that is part amylase containing extracts on

germination.

3. The function of HCl and NaOH in this practicum to influence if the solution

acid and base.