BIOCHEM LABORATORY

Exercise 4

ENZYMES

POST LABORATORY
ENZYMES
• Are soluble, colloidal, organic biocatalysts

• Speed up chemical reactions typically lowering the

activation of energy required for such reactions to
complete.

• They are also known as “ biological middleman”.

What we did?
• Amylase
• Iodine test
• Reactions before and after boiling the solution
• Benedict’s test?...

• Catalase
• Hydrogen peroxide actions toward
• Meat
• Leaf
• Potato

• Factors that affect enzyme activity

• Temperature?...
• pH?...
Action of Salivary
Amylase
 TUBE 1A
IODINE
TUBE 1A
STARCH
SOLN ONLY
HEAT TO
BOILING
TUBE 1B
STARCH
SOLN ONLY TUBE 1B

BENEDICT’S
REAGENT
 TUBE 2A

TUBE 2A
STARCH SOLN + IODINE
SALIVA
HEAT TO
40⁰C

TUBE 2B
STARCH SOLN + BENEDICT’S TUBE 2B
SALIVA REAGENT
 TUBE 1A

TUBE 1A IODINE
STARCH
SOLN ONLY
HEAT TO
40⁰C

TUBE 1B

TUBE 1B
STARCH
BENEDICT’S
SOLN ONLY REAGENT
Action of Salivary Amylase
IODINE BENEDICT’S REAGENT

1 2
1
 Principles behind the Test
• Amylase ( Iodine )

• If amylase is added to a solution of starch, it is expected to catalyze

the hydrolysis of starch to form into maltose, then with the
application of heat the faster the blue color of starch is lost, the
faster the enzyme amylase is working.

• No hydrolysis:
• Iodine does not stain maltose. When iodine is added to starch alone,
the solution turns blue. But if all the starch has been hydrolyzed to
maltose, the solution will not turn blue (it remains the yellowish color
of the iodine solution.
 Principles behind the Test
• Amylase (Benedicts)

• Amylases hydrolyzed the starch solution into smaller molecules-

being reduced , ultimately yielding maltose.

• The reducing sugars that show positive results with benedict's

solution are glucose, fructose, maltose.

• Complex carbohydrates such as starches DO NOT react positive with

the Benedict's test unless they are broken down through heating or
digestion.
Factors that affect enzyme
activity
TEMPERATURE
TUBE 1A

IODINE
HEAT TO
60⁰C

HEAT TO
100⁰C

TUBE 1B
BENEDICT’S
REAGENT
 TEMPERATURE
IODINE BENEDICT’S

1A 60⁰C Blue 1A 60⁰C Greenish

Blue

YELLOW 1A 100⁰C
1A 100⁰C Orange to
Red
TUBE 2A

IODINE
HEAT TO
60⁰C

HEAT TO
100⁰C

BENEDICT’S
TUBE 2B
REAGENT
 TEMPERATURE AFFECTS ENZYME ACTIVITY

IODINE BENEDICT’S

60⁰C Bluish 1A 60⁰C Blue with a

1A tinch of
Yellow
Orange

YELLOW 1A 100⁰C
1A 100⁰C Orange to
Red
TEMPERATURE AFFECTS ENZYME ACTIVITY
WITHOUT AMYLASE WITH AMYLASE
• In this laboratory experiment it also shows the factor
affecting enzyme activity. Particularly temperature.
• As temperature increases, enzyme activity also speeds up.
However, very high temperature can cause the enzyme to lose
its shape and stop working
• 40⁰C – Optimal temperature
• 60⁰C – reaction decrease sharply
• 100⁰C - enzyme is destroyed

• Changing the pH will slow enzyme activity.

• Extreme pH values also causes enzymes to denature.
Action of Catalase
Catalase test

Meat

Leaf
 Principles behind the Test
• Catalase

• You will see bubbling when the meat has the hydrogen peroxide
placed on it. That is because of the enzyme catalase.

• Catalase acts as the catalyzing enzyme in the decomposition of

hydrogen peroxide.

• Catalase decomposes or breaks down hydrogen peroxide into water

and oxygen
Thank you