International University - Vietnam National University

School Of Biotechnology

PRESENTATION
Topic: The role of cofactor in enzyme catalysis
The quantitative methods to determine amylase activity
Members:
Võ Thị Mỹ Duyên BTBTIU18330
Nguyễn Thị Quỳnh Giang BTBTWE18058
Nguyễn Hoàng Như Quỳnh BTBTWE18029
Dương Lê Thanh Thương BEBEIU19031
Nguyễn Ngọc Giang Thanh BTFTIU17073
Lê Hà Thu Uyên BTBTIU18351
Instructor: Dr. Le Hong Phu
OUTLINE

I. The role of cofactor in enzyme catalysis

II. The quantitative methods to determine amylase
activity
III. Summary
I. The role of cofactor in enzyme catalysis

1.Recalling Enzymes
2.Definition and classification of cofactors
3.Role of cofactors in enzyme catalysis
 1. Recalling Enzymes

Enzymes
 
Definition General role Manner

Aid in the
Protein composed Lowering the
performance of
of amino acids, energy of activation
biochemical
regardless of some without themselves
reactions to make
excuses being consumed.
life possible
1. Recalling Enzymes

The levels of
proteins in which
Enzymes are
fully functional
1. Recalling Enzymes

Almost any
  biochemical
reaction is made
possibly fast
enough to support
life thank to
biological catalysts.
 2. Definition and classification of cofactors

Definition
• A cofactor is a non-protein
chemical.
• Cofactors can be considered
"helper molecules" that assist
in biochemical transformations
 2. Definition and classification of cofactors

Classification
•Organic
•Inorganic
 3. Role of cofactors in enzyme catalysis

Organic cofactors (Coenzymes): -Can be reused and recycled without

-Activate enzyme: changing reaction rate or effectiveness
(like enzyme)
3. Role of cofactors in enzyme catalysis
Vitamins and derived from vitamins:
❖ Nicotinamide adenine
dinucleotide (NAD): A
coenzyme derived from
vitamin B3
3. Role of cofactors in enzyme catalysis
 3. Role of cofactors in enzyme catalysis
❖NAD+:
+ Works as a +Transfer electrons needed for redox reaction
substrate for DNA
ligases in
posttranslational
modification in
most bacteria
 3. Role of cofactors in enzyme catalysis

❖NADH (Coenzyme 1):

+ Carries electrons for
reactions and produces
energy from food
 3. Role of cofactors in enzyme catalysis
❖NADH (Coenzyme 1):
+ Anabolic reaction:
• Gluconeogenesis;
• Aids in the production of
neurotransmitters in the brain;
• Protecting cells against harmful or
damaging substances.
 3. Role of cofactors in enzyme catalysis
INORGANIC COFACTORS (Metal Cofactors): Plays a role as the
essential trace elements:

❖Mg2+: is used in glycolysis

❖Iron: is used in blood production
II. The quantitative methods to determine amylase activity

1.Definition and activation of amylase

2.Methods to determine amylase activity
3.Example of partical use
 1. Definition and activation of amylase

❖ DEFINITION
Are enzymes that catalyze the hydrolysis of
starch into smaller carbohydrate molecules.

❖ CLASSIFICATION
Base on differ in the way of attacking the bond of
starch molecules  Amylase includes: α-
amylase; β-amylase; Glucoamylase
 1. Definition and activation of amylase
❖ACTIVATION
  α-amylase β-amylase Glucoamylase
Activation Acts on starch related Acts on starch, glycogen and Cleaves the last α(1- 4)
polysaccharides and related polysaccharides and glycoside linkages non
oligosaccharides in a random oligosaccharides producing reducing end of a and
manner; reducing groups are β-maltose. amylopectin
liberated in the α-
configuration
The term β relates to the
initial anomeric configuration
of the free sugar group
released.

Source Can be found in digestive Is either produced by bacteria Can be found abundantly in
system of human and other or yeast or it can be found in fungi.
mammals, plants, microbes plants, particularly in the
seeds and grains.
 2. Methods to determine amylase activity

SOME METHODS DNS method

•Starch-iodine method •Introduction

• DNS method •Principle

•Reagent
• HPLC method
•Enzyme
• Nephelometric method
•Procedure
• Phadebas method
•Calculation
 2. Methods to determine amylase activity

DNS method
1/ Introduction:
• Estimatingthe concentration of reducing sugars
• Reducing sugars contain free carbonyl group
• All monosaccharide and some disaccharide
2/ Principle:

Low concentration High concentration

Intensity of the color is an 

index of reducing sugar
 2. Methods to determine amylase activity

DNS method
3/ Reagent: 4/ Enzyme:
• 0.02M Sodium phosphate buffer   •Alpha-amylase
•1% Starch  
•2N Sodium hydroxide  
•Sodium potassium tartrate tetrahydrate 
•Dinitrosalicylic acid color reagent  
•Standard Maltose Stock Solution  
•Amylase enzyme 
 2. Methods to determine amylase activity

DNS method
5/ Procedure: Adjust spectrophotometer at 540 nm and 25°C.
 2. Methods to determine amylase activity

DNS method
6/ Calculation:
3. Example of partical use

In
Medical
3. Example of partical use

❖Apply in diagnosis the diseases:

Assays of alpha
- amylase (AAS)
in serum and
urine by iodine
method.
Renal diseases
 3. Example of partical use

❖Advantages:
✔Only a one - step assay.
✔Limit the heavy financial.
✔Reduce the fee for lab
equipment.
✔Rapid and easy.
3. Example of partical use

In
Industry
3. Example of partical use

❖ Improve quality of food, product of crops.

 III. Summary
The role of cofactor in enzyme catalysis:
• Cofactor is a non-protein chemical. It can be considered "helper molecules" that assist in biochemical transformations.

• Cofactor includes 2 types:

+ ORGANIC: Vitamins and derived vitamin
NAD+
NADH (Coenzyme 1)

+ INORGANIC: Mg2+ - is used in glycolysis

Iron: is used in blood production

The quantitative methods to determine amylase activity

• Amylase are enzymes that catalyze the hydrolysis of starch into smaller carbohydrate molecules.

• Methods to determine amylase activity

+ Starch-iodine method
+ DNS method

• Application in Medical, Food Industry....

REFERENCES

• http://www.worthington-biochem.com/aa/assay.html
• https://www.cerealsgrains.org/publications/cc/backissues/1983/
documents/chem60_46.pdf
• https://pubs.rsc.org/en/content/articlelanding/2015/an/c4an02033b/
unauth#!divAbstract
• https://www.cerealsgrains.org/publications/cc/backissues/1964/
Documents/chem41_127.pdf
• Biochemistry, 5th edition, Jeremy M Berg, John L Tymoczko, and Lubert
Stryer