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

Mengapa Ezim Terisolasi ??

Untuk memahami sifat enzim:
Enzim bekerja dalam sistem yang kompleks
(dalam sel), == kita harus mencoba pertama
dalam sistem sederhana ==
Digunakan dalam makanan / aplikasi industri
- Pektinase: produksi Juice
- Chymosin: produksi keju
- Glukosa isomerase: produksi HFS

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

Mengapa enzim terisolasi??

Enzim terisolasi dapat dicirikan:
Kegiatan yang optimal dalam berbagai kondisi (pH,
temperatur, strengh ionik dll)
kekhususan
parameter kinetik
mekanisme katalisis
peraturan
struktur
Memahami peran enzim dalam sistem yang lebih
kompleks

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

Tujuan and Strategi

tujuan:

untuk mengisolasi enzim tertentu

dari semua protein lain (enzim)
dan komponen sel dengan
- Mungkin hasil maksimum
- Aktivitas katalitik maksimum
- Kemurnian maksimum yang
mungkin

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

Strategi
Protein yang beragam dalam perilaku

struktur komposisi, Anda harus tahu tentang

asal-usul mereka. Sebagai strategi
pemurnian Anda tergantung padanya.
1. Dimana enzim ini atau hadir protein dalam
sel? (intraseluler, ekstraseluler, membran).
2. Bagaimana Anda dapat memurnikan protein
ini sebagai beberapa langkah mungkin
tanpa kehilangan aktivitas.
3. Menjaga dalam pertimbangan suhu dan
waktu.

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

Sumber dari enzim

Mikroorganisme, hewan, tumbuhan

(jaringan dan sel)
-Rennin dari Mucor miehei
-Rennin dari lambung anak sapi

ekspresi heterolog
Bakteri rekayasa genetika, ragi untuk
memproduksi enzim
- Rennin / chymosin dari g.e. ragi
- Amiloglukosidase dari 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%)

Sumber dari enzim

Enzim industri dapat diekstraksi dari
setiap organisme hidup:

jamur (lebih dari setengah)

bakteri (lebih 3/1)
hewan (8%)
tanaman (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)

Sumber dari enzim

Mikroba yang lebih suka tanaman dan hewan
sebagai sumber enzim karena:

Mereka umumnya lebih murah untuk

memproduksi
Isi enzim mereka lebih dapat diprediksi dan
dikontrol
Jaringan tanaman dan hewan mengandung lebih
banyak bahan yang berpotensi berbahaya
daripada mikroba, termasuk senyawa fenolik (dari
tanaman)

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

Protokol umum untuk

pemurnian enzim
1.
2.
3.
4.
5.
6.
7.

8.

Mengambil jaringan sel utuh.

homogenisasi
Pemisahan puing sel dan hal-hal yang tidak
larut
Pengendapan protein dengan garam
Menyingkirkan garam dengan dialisis
Pemurnian lebih lanjut dengan kolom dan
kromatografi pertukaran ion,
Setiap langkah di atas diikuti oleh aktivitas
enzim assay (dalam kasus Anda kehilangan
enzim Anda)
Mencari tahu berat molekul yang tepat

Skema khas pemurnian enzim

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

Pengujian
pengembangan enzim
Bagaimana kita mengenali enzim yang kita

cari?

Mengembangkan uji analisis

- biasanya didasarkan pada reaksi bahwa

enzim
mengkatalisis dalam sel

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

Pemantauan Kemajuan Pemurnian 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

Homogenisasi dari
sampel
homogenisasi:

proses melanggar sel terbuka

Tergantung

pada sumber
- Jaringan mamalia
- Tanaman, jamur, bahan bakteri
- Ekstraksi enzim membran-terikat

Homogenisation
Methods
Physical

French pressure

cell
Sonication
Glass beads

Chemical
Detergents (SDS)
Enzymes

Homogenisation
Methods

fisik
-Sel tekanan
Perancis
-sonication
-manik-manik kaca

kimia
-Deterjen (SDS)
-Enzim (lyzozyme)
-penyangga
hipotonik

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

Fraksinasi sel /
Pemisahan
Pemisahan

puing sel dan hal-hal yang

tidak larut dapat difraksinasi dengan
sentrifugasi
Pada sentrifugasi, bahan padat akan
mengumpulkan di bagian bawah
tabung di pelet sedangkan bahan
dengan densitas yang lebih rendah
akan tetap dalam fraksi larut disebut
supernatan
Hanya sebagian kecil yang berisi

Cell Fractionation

Centrifugation

Supernatan

t
Pellet
Filtration

(other
methods)

Stabilize Sample ( stabilisasi

sampel)

Control pH
Use appropriate bufer

kontrol pH
-Gunakan penyangga yang tepat

kontrol suhu
Control temperature -Terus sampel di atas es atau
bekerja di ruangan yang dingin (0 Keep samples on ice or
4oC)
work in cold room (0 4oC)-instrumen Prechill

Prechill instruments

Prevent
frothing/foaming
Handle gently.

Maintain

Mencegah buih / busa

-Menangani dengan lembut.

Menjaga sampel terkonsentrasi

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
]

Solubilisasi protein
penggaraman di
-Pada konsentrasi rendah
garam, ion melindungi
biaya dan memungkinkan
protein melipat.
penggaraman
-Ion bersaing dengan air
untuk berinteraksi dengan
kelompok sisi.

-Ketika [garam] cukup

tinggi, garam menang

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)

Salting-in and Saltingout

Protein

yang berbeda endapan pada

konsentrasi garam yang berbeda,
sehingga konsentrasi garam dapat
disesuaikan untuk mengendapkan
protein yang diinginkan

Amonium

sulfat adalah digunakan

reagen yang paling umum untuk
penggaraman enzim (protein)

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

Suatu bentuk
kromatografi eksklusi
ukuran.

Digunakan untuk
menghilangkan garam
dan berkonsentrasi
sampel protein.

Berat molekul dialisis

tubing telah menetapkan
dipotong (MWCO). Hanya
molekul yang lebih kecil
dari MWCO akan

Dialysis

Column Chromatography
Most

common method for separating

enzymes and other proteins
Kebanyakan metode umum untuk
memisahkan enzim dan protein lain

Separating Proteins
(memisahkan protein

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.

Kromatografi
-fase gerak
-Fase yang membawa
sampel seluruh
prosedur.
-cair
-gas
fase stasioner
-Matriks yang
menghambat pergerakan
sampel yang dibawa oleh
fase gerak.

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

Juga disebut kromatografi eksklusi ukuran

Enzim dipisahkan atas
dasar ukuran.

Molekul besar keluar

pertama.

fase gerak
-cair

fase stasioner
-Larut, manik-manik
karbohidrat berpori

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

Gel-filtration
Chromatography
Sampel diterapkan ke atas kolom yang berisi
berpori manik / matriks
Molekul kecil akan melewati pori-pori manikmanik (sementara yang lebih besar tidak bisa)
Molekul besar mengalir lebih cepat melalui
kolom dan terelusi pertama
Molekul ukuran menengah akan mengelusi di
posisi menengah (kadang-kadang memasuki
manik-manik)
Molekul-molekul kecil akan mengelusi lalu,
karena mengambil jalan lagi

Ion Exchange
Chromatography

Memisahkan
Separates
molekul
molecules based
berdasarkan biaya.
on charge.
fase gerak
Mobile phase

H2
C
cellulos
e

O
C

-umumnya cairan
fase stasioner
O
Stationary phase
-Elektrostatis
Electrostatically
carboxymethyl(CM)
dibebankan ion terikat
charged ions
(Cation
larut, matriks kimia
bound to
exchange)
inert.
insoluble,
CH3
chemically inert Elusi protein
H2C
-Tambahkan garam
H
matrix.
H2
untuk bersaing dengan
N
CH3
Elution of
C
C
C
pengikatan sampel cellulos
protein
H2
H2
untuk
fase
diam.
e
Add salt to
-Perubahan pH (alter
diethylaminoethyl(DEAE)
compete with
(Anion
binding of sample biaya protein).
Generally liquid

Ion Exchange
Chromatography

Ion exchange resins contain charged groups.

Resin pertukaran ion mengandung gugus
dikenakan.

If these groups are acidic in nature they

interact with positively charged proteins are
called cation exchangers.
Jika kelompok-kelompok ini bersifat asam di
Positively
CH2-COO- +
alam mereka berinteraksi
protein
+ dengan
charged
(basic)
CH2-COO
+ kation
protein
or
bermuatan positif
disebut
exchanger.
+

CM cellulose
cation exchanger

enzyme

Ion Exchange
Chromatography
If these groups are basic in nature, they
interact with negatively charged molecules are
called anion exchangers.
Jika kelompok-kelompok ini dasar di alam,
mereka berinteraksi dengan molekul
bermuatan negatif disebut -penukar
anion.
Negatively

CH2-CH2 -NH+(CH2CH2)

CH2-CH2 -NH+(CH2CH2) DEAE cellulose

anion exchanger
DEAE selulosa
anion exchanger

charged
(acidic) protein
or enzyme
Bermuatan
negatif (asam)
protein atau
enzim

Ion Exchange
Chromatography
For protein binding, the pH is fixed (usually near neutral)
under low salt conditions. Example cation exchange
column
Untuk protein yang mengikat, pH adalah tetap (biasanya
mendekati netral) dalam kondisi rendah
garam.
Misalnya
Positively
charged
+
tukar kation kolom
CH...
-COO
+
2
protein or enzyme bind
CH2-COO- +
+
CM cellulose
cation exchanger

to the column
Bermuatan positif
protein atau enzim
mengikat ke kolom
Negatively charged
proteins pass through
the column
Protein bermuatan
negatif melewati
kolom

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.
Untuk mengelusi enzim yang menarik, tambahkan jumlah yang
semakin tinggi garam (meningkatkan kekuatan ion). Na + akan
berinteraksi dengan resin kation dan-+Cl- akan berinteraksi dengan
+ Increasing
CH2-COO +
protein kami bermuatan positif untuk mengelusi dari kolom.
[NaCl] of the
CH2-COO- +
elution bufer
+
CM cellulose
cation exchanger
Cl+
ClCH2-COO- Na+ Na+2
+
CH2-COO Na+
Cl
Na+2
+
CM cellulose
cation exchanger

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
Kromatografi afinitas adalah metode memisahkan campuran enzim / protein
didasarkan pada interaksi yang sangat spesifik antara: antibodi dan antigen
substrat dan enzim
ligan dan reseptor

Mobile phase
Usually liquid

Stationary
phase
Antibody/substra

te/ligand bound

fase gerak
biasanya cairan
fase stasioner
Antibodi / substrat /
ligan terikat manik
lembam

Immunoaffin
ity

Affinity chromatography

Affinity chromatography
Possible

elutionKemungkinan
Pengembangan strategi
strategies:

elusi:
pH
ion strengh
mengubah Sifat Sesuatu
Benda
Competitor ligand or
Pesaing ligan ATAU
analog
analog

pH
Ion strengh
Denature

Ab affinity column

Monitoring Progress of
Purification Protocol
Total

protein (mg)

Quantity of protein

present in fraction

Jumlah protein (mg)

Jumlah protein hadir dalam
fraksi

Total

activity (units
Jumlah aktivitas (unit
of activity)
kegiatan)

Use a portion of sampleMenggunakan sebagian

to determine activity. dari sampel untuk
menentukan aktivitas.
Multiply activity by total
Kegiatan kalikan dengan
volume to determine total volume untuk
total activity.
menentukan aktivitas total.

Monitoring Progress of
Purification Protocol

Specific activity (units of activity/mg)

S.A. =Total activity
Total protein

% yield: measure of activity retained

after each step in procedure
ukuran aktivitas ditahan setelah setiap
Total activity at particular
%
yield
=
langkah dalam prosedur.
step

Total activity of initial extract

Monitoring Progress of
Purification Protocol

Purification level: Measure of

increase in purity of protein throughout
procedure.

Tingkat pemurnian: Mengukur peningkatan

kemurnian protein seluruh prosedur.

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. Complete mix
proteins
2. High Salt
3. Ion exchange
4. Gel-filtration
5. Affinity

of1. Campuran
lengkap
protein
2. Salt tinggi
3. pertukaran ion
4. Gel-filtrasi
5. afinitas

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
o Molecular mass

massa molekul
o Optimum activity

Kegiatan optimal
in various conditions
dalam berbagai kondisi
(pH, temperatur, (pH, temperatur, ion
ionic strengh etc)
strengh dll)
o Enzyme

spesifisitas enzim
specificity
parameter kinetik
o Kinetic

Mekanisme katalisis
parameter

Struktur (X-ray
o Mechanism of
kristalografi)
catalysis

peraturan
o Structure (X-ray

Electrophoresis

Separates
molecules based
on molecular
mass and/or
charge.
Memisahkan
molekul
berdasarkan
massa molekul
dan / atau biaya

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

SDS-PAGE

Sodium dodecyl Sodium dodecyl

sulfat poliakrilamida
sulfate
gel elektroforesis
polyacrylamide
gel
electrophoresis Pemisahan

berdasarkan massa
molekul.

Separation
based on
Sampel mantel
molecular mass.
dengan SDS untuk
Coat samples
with SDS to give
uniform charge
to mass ratio.
Makes all

memberikan biaya
seragam untuk
rasio massa.
Membuat semua
protein bermuatan
negatif.

Protein separation using SDS-PAGE

(Laemmli system)

1.

Apply protein/dye samples

into polyacrylamide gel
wells
Terapkan sampel protein / dye?
dalam sumur gel poliakrilamida

Stacking
gel

Resolving
gel

3. Remove the gel from

the apparatus and stain
for proteins
Lepaskan gel dari? alat
dan noda untuk? protein

2. Run the electrophoresis until dye

reaches the end of the gel
Jalankan elektroforesis sampai dye?
mencapai akhir gel

Isoelectric Focusing

Separation based on charge.

Polyampholite (small
Pemisahan berdasarkan biaya.
multicharged polymers) used to Polyampholite (polimer
prepare pH gradient in the gel
multicharged kecil) digunakan untuk
Can be used to experimentally
mempersiapkan pH gradien dalam
gel
determine the pI values.
Dapat digunakan untuk eksperimen
menentukan nilai pI.

THANK YOU