Mata Kuliah Biokimia

- MKK4213
STF MUHAMMADIYAH

PEMURNIAN
ENZIM

Why isolate enzymes??

To

understand enzyme properties:

Enzymes work in a complex system
(inside cell),
== we must try first in
a simple system ==

Used

in food / industrial
applications
- Pectinase : Juice production
- Chymosin : cheese production
- Glucose isomerase : HFS production

Why isolate enzymes??

The isolated enzyme can be
characterized:
o optimum activity in various conditions

(pH, temperatur, ionic strengh etc)

o specificity
o kinetic parameter
o mechanism of catalysis
o regulation
o Structure
Understand

the role of enzymes

How to Separate These Objects

Shape
Size
Density

2 3 4 5 6

10 11

12

wood stone cotton wood wood cotton stone wood stone cotton stone cotton

2 3

Shape

Size

4 5 6

7 8
Density

7
9

7 8 9

10 11

cotton

4
8

wood

stone

12

Sieving different sizes

Different sedimentation

Different rolling speed

Juang RH (2004) BCbasics

Objectives and Strategy

Objectives:

to isolate a particular enzyme from

all other proteins (enzyme) and cell
components with
- maximum possible yield
- maximum catalytic activity
- maximum possible purity

Objectives and Strategy

Strategy
:
Material

Homogenisation

Whole
extraction

Large scale separation

Affinity
Separatio
n

Crude extract

Series of
small
scale
separatio
n

Pure
enzyme

Strategy
Protein

are diverse in composition

structure behaviour, you should know
about their origin. As your purification
strategy depends on it.
1. Where is this enzyme or protein present in

the cell? (intracellular, extracellular,

membranous).
2. How you can purify this protein in as few
steps as possible without the loss of
activity.
3. Keeping in consideration of temperature and

Sources of Enzymes

Microorganism, animal, plant (tissue

and cell)
Rennin from Mucor miehei
Rennin from stomach of calf

Heterologous expression
Genetically engineered bacteria, yeast
to produce enzyme
- Rennin / chymosin from g.e. yeast
-

Amyloglucosidase from g.e. Bacillus

licheniformis

Sources of Enzymes
Industrial enzymes may be
extracted from any living organism:
fungi (over a half)
bacteria (over 1 / 3)
animal (8%)
plant (4%)

Sources of Enzymes
Microbes are preferred to plants and
animals as sources of enzymes because:

They are generally cheaper to produce

Their enzyme contents are more predictable and
controllable
Plant and animal tissues contain more potentially
harmful materials than microbes, including
phenolic compounds (from plants)

Fungal Enzymes
Enzyme

EC

Sources

Application

-Amylase

3.2.1.1

Aspergillus

Baking

Catalase

1.11.1.6

Aspergillus

Food

Cellulase

3.2.1.4

Trichoderma

Waste

Dextranase

3.2.1.11

Penicillium

Food

Glucose oxidase

1.1.3.4

Aspergillus

Food

Lactase

3.2.1.23

Aspergillus

Dairy

Lipase

3.1.1.3

Rhizopus

Food

Rennet

3.4.23.6

Mucor miehei

Cheese

Pectinase

3.2.1.15

Aspergillus

Drinks

Protease

3.4.23.6

Aspergillus

Baking

E: extracellular enzyme; I: intracellular enzyme

Bacterial Enzymes
Sources

Enzyme

Application

-Amylase

3.2.1.1

Bacillus

Starch

-Amylase

3.2.1.2

Bacillus

Starch

Asparaginase

3.5.1.1

Escherichia coli

Health

Glucose isomerase

5.3.1.5

Bacillus

Fructose syrup

Penicillin amidase

3.5.1.11

Bacillus

Pharmaceutical

Protease

3.4.21.14

Bacillus

Detergent

Basic Principles of Protein (Enzyme) Purification

Cell

Homogenization

Macromolecule

Small molecule
Amino acid, Sugar,
Nucleotides, etc

Size
Gel filtration,
SDS-PAGE,
Ultrafiltration

Organelle

Nucleic
acid

Protein

Charge
Ion exchange,
Isoelectric focusing

Carbohydrate

Solubility
Salting-out
(Ammonium
sulfate )

(Lipid)

Cell
Debris

Affinity
Affinity
chromatography

General protocol for enzyme

purification
1.
2.
3.
4.
5.
6.
7.
8.

Taking the intact cell tissue.

Homogenisation
Separation of cell debris and insoluble stuf
Precipitation of protein with the salt
Getting rid of salt by dialysis
Further purification by column and ion
exchange chromatography ,
Each above step is followed by enzyme assay
activity(in case you lost your enzyme )
Finding out the exact molecular weight by
column chromatography and by SDS-Gelelectrophoresis

Typical Enzyme Purification

Develope Enzyme Assay

How do we recognize the enzyme

that we are looking for?

Develop analytical assay

usually based on the reaction that the enzyme

catalyzes in the cell

Develope Enzyme Assay

OO

N+

HO

HO

OH

OH
O

OH

O
OH

OH

ONPG

galactosidase

OH

O
OH

N+
O-

OH

Galactose

ONP

Monitoring Progress of
Purification Protocol
Step

Total protein
(mg)

Total activity
(units)

Specific
activity
(units/mg)

Yield
(%)

Purification
level (Purity
factor)

Initial extract

15,000

150,000

10

100

(NH4)2SO4
precipitation

4,600

138,000

30

92

Ion-exchange

1,278

115,500

90

77

Size
exclusion

68.6

75,000

1,100

50

110

Affinity
column

1.75

52,500

30,000

35

3,000

(Berg, Tymoczko, & Stryer. (2002) Biochemistry, 5th ed. W.H. Freeman & Co., New York, NY,
p. 86)

PURIFICATION METHODS

Homogenisation of
sample
Homogenisation

:
the process of breaking open
cells

Depend

on the source
- Mammalian tissue
- Plant, fungal, bacterial material
- Extraction of membrane-bound
enzymes

Homogenisation
Methods
Physical

French pressure

cell
Sonication
Glass beads

Chemical
Detergents (SDS)
Enzymes

Cell Fractionation /
Separation

Separation of cell debris and insoluble

stuf can be fractionated by
centrifugation

In centrifugation, denser material will

collect at the bottom of the tube in a
pellet whereas material with lower
density will remain in the soluble
fraction called the supernatant

Only the fraction that contains the protein

Cell Fractionation

Centrifugation

Supernatan

t
Pellet
Filtration

(other
methods)

Stabilize Sample

Control pH
Use appropriate bufer

Control temperature
Keep samples on ice or work in cold room (0

4oC)
Prechill instruments

Prevent frothing/foaming
Handle gently.

Maintain concentrated sample

Stabilize Sample
Protease

inhibitors

Phenylmethylsulfonyl

fluoride (PMSF)
O
S
O
PMSF

Protein Solubility
Salting in
At a low salt
concentration, ions
protect charges and
allow proteins to fold.

Salting out
Ions compete with water
to interact with side
groups.
When [salt] is high

enough, salt wins

solubili
ty

salting in

salting
out

[salt
]

Salting in / salting out

Salting-in and Saltingout

Diferent

proteins precipitate at
diferent salt concentrations, so the
salt concentration may be
adjusted to precipitate the
desired protein

Ammonium

sulfate is the most

commonly used reagent for salting
out enzymes (proteins)

Dialysis

A form of size exclusion

chromatography.

Used to desalt and

concentrate protein
samples.

Dialysis tubing has set

molecular weight cut
off (MWCO). Only
molecules that smaller
than MWCO will move
out of the dialysis bag.

Dialysis

Column Chromatography
Most

common method for separating

enzymes and other proteins

Separating Proteins

Chromatography
Mobile phase
Phase that carries sample
throughout procedure.
Liquid
Gas
Stationary phase
Matrix that retards the movement of
sample being carried by the mobile
phase.

Gel-filtration
Chromatography
Also

called size exclusion

Enzymes are separated on
chromatography
the basis of size.

Large molecules exit first.

Mobile phase
Liquid

Stationary phase
Insoluble, porous
carbohydrate beads

Gel-filtration
Chromatography

Gel-filtration
Chromatography

Sample is applied to the top of a column

containing porous beads / matrix

Small molecules will pass through the pores of

the beads (while larger ones cannot)
Large molecules flow more rapidly through the
column and elute first
Intermediate size molecules will elute at an
intermediate position (occasionally enter the
beads)
The small molecules will elute last, because
take longer path

Ion Exchange
Chromatography

Separates molecules based

on charge.
Mobile phase

H2
C

O-

carboxymethyl(CM)
(Cation
exchange)

Stationary phase
Electrostatically charged ions

bound to insoluble, chemically

inert matrix.

Elution of protein
Add salt to compete with binding

of sample to stationary phase.

Change pH (alters charge of

cellulos
e

Generally liquid

H2C
H2
C
cellulos
e

CH3
H

N
C
H2

C
H2

CH3

diethylaminoethyl(DEAE)
(Anion

Ion Exchange
Chromatography

Ion exchange resins contain charged groups.

If these groups are acidic in nature they

interact with positively charged proteins are
called cation exchangers.

CH2-COOCH2-COO

CM cellulose
cation exchanger

+
+
+

Positively
charged (basic)
protein or
enzyme

Ion Exchange
Chromatography

If these groups are basic in nature, they

interact with negatively charged molecules are
called anion exchangers.

CH2-CH2 -NH+(CH2CH2) CH2-CH2 -NH+(CH2CH2)

DEAE cellulose
anion exchanger

Negatively
charged
(acidic) protein
or enzyme

Ion Exchange
Chromatography
For protein binding, the pH is fixed (usually
near neutral) under low salt conditions.
Example cation exchange column
+
CH2-COO- +
CH2-COO- +
+
CM cellulose
cation exchanger

Positively charged
protein or enzyme bind
to the column

Negatively
charged
proteins pass
through the
column

Ion Exchange
Chromatography
To elute enzyme of interest, add increasingly higher
amount of salt (increase the ionic strength). Na + will
interact with the cation resin and Cl- will interact with
our positively charged protein to elute of the column.
CH2-COO-+ +
CH2-COO- +
+

CM cellulose
cation exchanger

CH2-COO- Na+ Na+2

CH2-COO- Na+
CM cellulose
cation exchanger

Na+2

+ Increasing
[NaCl] of the
elution bufer

ClCl- +
+
Cl+
Cl- +

Ion Exchange
Chromatography

Low salt

High salt

Examples
Name

Ionizable group

Type

DEAE-Sephadex

Diethylaminoethyl Weakly basic

SP-Sepharose

Methylsulfonate

Strongly acidic

Bio-Rex 70

Carboxylic acid

Weakly acidic

P cellulose

Phosphate

Strongly & weakly

acidic

Affinity Chromatography
Affinity chromatography is a method of separating
enzyme / protein mixtures based on a highly specific
interaction between : antibody and antigen
substrate and enzyme
ligand and receptor

Mobile phase
Usually liquid

Stationary phase
Antibody/substrate/ligand

bound to inert bead

Immunoaffin
ity

Affinity chromatography

Affinity chromatography
Possible

elution strategies:

pH
Ion strengh
Denature
Competitor ligand or analog

Ab affinity column

Monitoring Progress of
Purification Protocol
Total

protein (mg)

Quantity of protein present in fraction

Total

activity (units of activity)

Use a portion of sample to determine

activity.
Multiply activity by total volume to

determine total activity.

Monitoring Progress of
Purification Protocol

Specific activity (units of activity/mg)

S.A. =Total activity
Total protein

% yield: measure of activity retained

Total
activity at particular
after each
step
in procedure.
% yield
=

step
Total activity of initial extract

Monitoring Progress of
Purification Protocol

Purification level: Measure of

increase in purity of protein throughout
procedure.

Purification
level =

Specific activity at particular step

Specific activity of initial extract

Monitoring Progress of
Purification Protocol
Step

Total protein
(mg)

Total activity
(units)

Specific
activity
(units/mg)

Yield (%)

Purification
level

Initial extract

15,000

150,000

10

100

(NH4)2SO4
precipitation

4,600

138,000

30

92

Ion-exchange

1,278

115,500

90,3

77

9,03

Size
exclusion

68.6

75,000

1093,3

50

109,3

Affinity
column

1.75

52,500

30000

35

3000

(Berg, Tymoczko, & Stryer. (2002) Biochemistry, 5th ed. W.H. Freeman & Co., New York, NY,
p. 86)

SDS PAGE of Purification

1.
2.
3.
4.
5.

Complete mix of proteins

High Salt
Ion exchange
Gel-filtration
Affinity

10micrograms loaded in each lane

Purification Summary
STEP

Total
Activity
(Unit)

Total
Protein
(mg)

Specific
Activity
(Unit/mg)

Yield
(%)

Purification
factor (Fold)

Culture Filtrate

39.4

203

0.19

100

Colloidal Chitin Affinity

8.00

7.08

1.12

20.3

5.89

DEAE-Toyopearl M 650

6.45

4.11

1.57

16.3

8.21

Butyl-Toyopearl M 650

1.15

0.28

4.21

2.91

22.15

(kDa) M

Ra-ChiA
97
67

Ra-ChiA

AD PY LK VAYYP

Ba-ChiA1 A D S Y

K I V D YYP

N-terminal amino acid sequences

alignment of Ra-ChiA and Bc-ChiA1

43

30

SDS-PAGE

ZYMOGRAM

Characterization of
Enzyme
Molecular mass
o Optimum activity in various
conditions
(pH, temperatur, ionic strengh etc)
o Enzyme specificity
o Kinetic parameter
o Mechanism of catalysis
o Structure (X-ray crystallography)
o Regulation
o

Electrophoresis

http://www.science.fau.edu/chemistry/Mari/biochemlab/manual.html

Separates
molecules based
on molecular
mass and/or
charge.

SDS-PAGE

Sodium dodecyl sulfate

polyacrylamide gel
electrophoresis

Separation based on
molecular mass.

Coat samples with SDS

to give uniform charge to
mass ratio.
Makes all proteins

Protein separation using SDS-PAGE

(Laemmli system)

1. Apply protein/dye samples

into polyacrylamide gel wells

Stacking
gel

Resolving
gel

3. Remove the gel from the

apparatus and stain for
proteins

2. Run the electrophoresis until dye

reaches the end of the gel

Isoelectric Focusing

Separation based on charge.

Polyampholite (small multicharged polymers) used
to prepare pH gradient in the gel
Can be used to experimentally determine the pI
values.

THANK YOU