eKsTraKsI dan

PuriFikaSi
A. Pendahuluan
Beberapa teknik isolasi enzim secara umum
dikelompokan menjadi:
Metode klasik berupa distilasi dan ekstraksi dengan
pelarut organik (berdasarkan sifat umum makromolekul:
pH, kekuatan ion dan kelarutan) Kurang digunakan lagi
karena kaitannya dengan kestabilan enzim
Perbedaan sifat protein globular: Mr, muatan protein
Interaksi spesifik dan reversibel antara enzim dengan
substrat, koenzim, ligan
Penemuan separasi enzim
Penemuan
Pengendapan dengan alkohol
Adsorpsi spesifik amylase dengan
substrat insolubel
Penggunaan adsorbant untuk
pemurnian enzim
Penggunaan ultrasentrifugasi
pertama kali
Kristalisasi Urease
80 enzim terisolasi
Pengenalan resin penukar ion
Tahun
1833 (Payen & Persoz)
1910 (Starkenstein)
1922 (Willstarter)
1923 (Svedberg & Nichols)
1926 (Summer)
1930
1935 (Adams & Holmes)
Kromatografi adsorpsi
Metode Pengendapan terfraksi
dengan pelarut organik
Kromatografi dengan hidroksi
apatit
Pengenalan penukar ion selulosa
Pengenalan sepadex dan gel
filtrasi
Elektroforesis Protein
Introduksi aktivasi dengan CNBr
SDS Page elektroforesis
Induksi konsep kromatografi
afinitas
Kromatografi hidrofob
Lebih dari 2000 enzim terisolasi
1938-1941 (Zechmeister &
Brockman)
1946 (Cohn)
1951-1956 (Tiselius & Swingle)
1956 (Sober & Peterson)
1959 (Porath & Flodin)
1966 (Vesterberg)
1967 (Axen & Porath)
1967 (Shapiro)
1968 (Cuatrecasas)
1971 (Yon, Er El & Shaltiel)
1980
PEMISAHAN MATERIAL

Pengambilan bahan tidak larut (Removal of Insolubles). Sedikit

mengkonsentrasikan produk atau perbaikan produk. Filtrasi dan
sentrifugasi.
Isolasi Produk. Tidak spesifik, pengambilan bahan yang mempunyai
sifat yang tersebar dibandingkan dengan produk yang diinginkan.
Konsentrasi dan kwalitas produk mulai terjadi. Adsorpsi dan
ekstraksi solven.
Purifikasi. Teknik proses yang sangat selektif untuk menghasilkan
produk dan mengambil bahan yang tidak diinginkan serupa dengan
fungsi kimia dan sifat fisika. Khromatografi, elektrophoresis, dan
presipitasi.
Produk akhir. Kristalisasi
B. Tahapan isolasi
Lokasi enzim:
Ekstraseluler (ekoenzim)
Endoseluler (terikat pada partikel
subseluler atau membran sel)
Hal yang perlu diperhatikan
Sifat khas materi utama
Bentuk (cair, padat)
Tipe umum (animal, vegetal, mikroba)
Struktur biologi (seluler, tisuler)
Lokasi produk yang dicari: mitokondria,
sitoplasma, membran, dll.
 Sifat struktural dan fisiko-kimia:
Mr, struktur molekul, stabilitas
pH optimum, pI
Aktivator, inhibitor
Konstanta kinetika
Pelepasan protein dalam bentuk cair tanpa
menghilangkan aktivitas
Tanpa merusak struktur
Temperatur rendah (4c)
Penggunaan buffer
Penggunaan reagen pelindung (EDTA, 2-mercaptoethanol,
substrat, dll
Perlakuan secara cepat dan hati-hati
B.1. Tahapan Isolasi
Materi utama sangat heterogen, maka untuk
mendapatkan enzim murni perlu tahapan isolasi
Ekstraksi: peelepasan enzim dari sel atau bagian sel dan
didapatkan ekstrak dalam bentuk cair yang mempunyai
sifat fisiko kimia sama
Fraksionasi: memisahkan ekstraks berdasarkan
kelarutannya guna mendapatkan kelompok molekul yang
sama (fraksi)
Purifikasi: pemisahan fraksi lebih lanjut dengan metode
fisiko-kimia atau biospesifik untuk mendapatkan molekul
enzim lebih murni
Skema umum isolasi dan
purifikasi enzim
Materi Primer

Animal Vegetal Mikroba

Tahap Ekstraksi
Ekstrak total
Tahap Fraksionasi
Ekstrak kasar
Tahap Purifikasi

Enzim murni
Microbial source
Often more stable than analogous enzyme obtained from
plant or animal tissue
Generally Recognized As Safe (GRAS) certified microbes are
non pathogenic, nontoxic, and generally they do not produce
antibiotics

Bacteria Bacillus subtilis

B. Amyloliquifaciens
L. spesies
Fungi Aspergillus sp.
Penicillium sp.
Mucor
Rhizobium
S. cerevesiae
Plant source
Represent a traditional source of a wide range of enzymes
Plant tissues are chosen as a source for subsequent
purification of various enzymes

Enzyme Plant source

Ascorbate oxidase Curcubita species
Urease Jack bean
Bromealin Pineapple stem/fruit
Amylase Barley
Pectine esterase Citrus fruits
Phytase Wheat,rye, triticale
Animal source
Animal tissues are a source of several enzymes of industrial
use and therapeutic use
The other organs like stomach, placenta, heart, kidney or cells
like erythrocytes can be sources for specific enzymes

Enzyme Animal source

Acetyl choline esterase Bovine erythrocytes
Arginase Beef liver
Creatine kinase Rabbit muscle, beef heart
Aldose reductase Beef eyes
Uricase Porcine liver
Trypsin Mammalian pancreas
 STEP IN ENZYME PURIFICATION
Step Target Treatment
I Choosing enzyme source and Filtration
recovery Centrifugation
Enzyme Cell disruption
extract II Removal of whole cells and cell Centrifugation
debris from enzyme extract Filtration
Crude
III Removal of nucleic acids and Precipitation
Enzyme
lipids Nucleases
Dilute Glass wool
Enzyme
IV Concentration and primary Ultrafiltration
purification Precipitation
Chromatography
Conc. Dehydration
Enzyme V Final purification and quality Gel filtration
check Ion exchange
Affinity
Hydrophobic interaction
Chromatofocussing
HPLC
 Profil Proses
Tingkatan Produk
Proses Kons. (g/l) Kwalitas (%)
Pemanenan Fermentasi 0.1 5 0.1 1.0

Pengambilan bahan Filtrasi 1.0 5 0.2 2.0

tidak terlarut
Isolasi Ekstraksi 5 50 1 10

Purifikasi Kromatografi 50 200 50 80

Produk akhir Kristalisasi 50 200 90 100

Teknik separasi dan purifikasi berdasarkan sifatnya
Ultrasentrifugasi
Dialisa Kromatografi
Gel Filtrasi penukar ion Elektroforesis

Ultrasentrifugasi
Sentrifugasi zonal Gel Elektroforesis Mobilitas elektroforesis

Mr Muatan
Elektro
Densitas Enzim Titik isoelektrik dekantasi

Sifat permukaan Kelarutan

Stabilitas
Pengendapan
Kromatografi Isoelektrik
Adsorpsi Perlakuan :
Kromatografi: asam-basa Partisi
Afinitas suhu Cair-cair
Hidrofobik Pengendapan terfraksi
Kovalen dengan garam atau
pelarut organik
B.2. Kontrol Kualitas
Enzim industrial perlu adanya kontrol kualitas yang
meliputi;
Kemurnian enzim dalam periode waktu tertentu (aktivitas
spesifik)
Kontrol kemurnian dengan metode fisiko-kimia;
homogenitas dan sifat karakteristiknya (Mr,
polimorfisme...)
Uji stabilitas: resiko denaturasi, semakin murni enzim
semakin mudah terdenaturasi. Perlu dilakukan:
Eliminasi kontaminan
Penyimpanan pada temperatur renadah
pH netral
 Prosedur umum kontrol kwalitas enzim murni
Pengukuran :
Elektroforesis
Aktivitas katalitik
Ultrasentrifugasi Pengukuran:
Protein
Gel Filtrasi Tekanan osmose
Aktivitas spesifik
Kontaminan (enzim & Pengendapan dengan UF
lainnya Difusi dan koefisien difusi
Gel eksklusi kromatografi
Aktivitas biologi
& Spesifik Homogenitas SDS PAGE
Mr
Enzim murni Metode Sanger
Polimorfisme, Mr
Komposisi & Sequence
Kemasan
Stabilitas
Label Penggunaan
C. Ekstraksi
Ekstraksi; pelepasan enzim dari sel atau
bagian sel menggunakan proses mekanik,
dan non mekanik (kimia, enzimatis, dll)
Jaringan vegetal dan animal: penghalusan
dan homogenisasi, secara mekanik
Sel mikroorganisme secara umum adalah
pemecahan dinding sel secara mekanik dan
non mekanik
Kimia: alkali/asam, deterjen, osmose, EDTA
memecah bakteri gram negatif
Enzimatis: lisosim (memutus 1-4 glukosida
peptidoglican)
 Metode ekstraksi
Metode Perlakuan
Pemecahan jaringan dan Mekanik : potong, pecah dan homogenasi
sel Osilasi frekwensi tinggi: ultrasonikasi, turmix
Grinding
Pemecahan dan homogenisasi dengan tekanan
tinggi
Ekstraksi dengan pelarut Temperatur : shock dingin
air pH : shock alkali atau asam
Konsentrasi garam : shock osmosis
Efek spesifik substrat
Metode ekstraksi spesial Pelarut organik: butanol, aseton dan pelarut lipid
Pembekuan dan pencairan
Penggunaan deterjen Na-deoksi kolat, Tween 20, Teepol XL, Triton X-
100
Ekstraksi dengan enzim Otolisis: proteolisa dan lipolisa
Penggunaan enzim pemurni (tripsin, lipase)
Pemecahan dinding mikroorganisme
Proses mekanik:
Ultrasonikasi: sel dipecah
Pembekuan-pencairan
Penggerusan/agitasi dengan partikel gelas
Desintegrasi pada P>
Proses non-mekanik:
Desikasi dengan spray drying
Liase secara kimia dan fisika
Perlakuan alkali
Deterjen: Na-lauril sulfat, trixtron X-100
Shock dingin : jumlah kecil
Shock osmotic: perubahan konsentrasi garam
Liase Enzimatik
Lisozim : hidrolisis beta 1,4 glukosida
Autolisis: dengan proteolise, lipolise
D. FRAKSIONASI
D.1. FRAKSIONASI PENGENDAPAN
D.1.1. PENGENDAPAN DENGAN GARAM
Garam yang paling banyak digunakan Amm.
Sulfat
Prinsip:
Protein larut dalam larutan garam pada pH
sekitar pI
Kelarutan lebih kuat dibanding dengan kekuatan
ion dalam larutan (salting in)
Batas kekuatan ion tertentu, kelarutan
berkurang (salting out) berkaitan dengan
terhidrasinya protein
 Daerah pengendapan bergantung pada:
Garam yang digunakan
Jenis proteinnya
Kelebihan (NH4)2SO4:
Harganya murah
Kemampuan pengendapan tinggi
Kelarutannya besar, endotermik
Efek denaturasi terhadap protein rendah
Kristalisasi garam tertentu yang
terendapkan oleh konsentrasi garam
tertentu dapat dilarutkan kembali
dengan melarutkannya pada pelarut
dengan kadar lebih rendah
D.1.2. PENGENDAPAN PADA ISOELEKTRIK
pI Kelarutan minimum, mengendap

Pengaturan pH larutan

D.1.3. EFEK TEMPERATUR

Protein Globular T>Kelarutan>, s/d 40-50C

Hemoglobin

Pengaturan T Seleksi Protein

Protein terdenaturasi

Tidak dapat digunakan

secara industri
 D.1.2. PENGENDAPAN DENGAN PELARUT
ORGANIK
Enzim Protein Protein
Saling bergabung mengendap

<: konstanta dielektrikum

& kestabilan

Tambah pelarut organik Penambahan pelarut pada

T< shg tidak mendenaturasi
Alkohol
Isopropanol (paling banyak digunakan) untuk enzim
ekstraseluler; amiloglukosidase
Methanol
Aseton dan etil eter: protein sedikit larut maka perlu
jumlah yang banyak
 Variable Extraction Adsorption

Capacity High Low

Selectivity Moderate High

Nature of equilibrium Often linier; Usually non linier; dilute

dilute solutes solutes interact
independent
Nature of operation Steady state Unsteady; periodic
Problems Emulsification; Solids handling;
denaturation compressible packing
Electrophoresis
Electrophoresis
Principle is to separate proteins (in
tact) on the basis of their charge and
their ability to migrate within a gel
(jello-like) matrix
A strong electric field is applied to the
protein mixture for an extended
period of time (hours) until the
proteins move apart or migrate
Isoelectric Focusing (IEF)
 Isoelectric Point (pI)
The pH at which a protein has a net charge=0
Q = S Ni/(1 + 10pH-pKi)
Transcendental
equation

pKa Values for Ionizable Amno Acids

Residue pKa Residue pKa

C 10.28 H 6
D 3.65 K 10.53
E 4.25 R 12.43
IEF Principles
Increasing pH

+ _
_ C
A + A
+ _
N _ T
O + H
+ _
D _ O
E + D
+ _
_ E
+
+ _

pI = 5.1 pI = 6.4 pI = 8.6

 Isoelectric Focusing
Separation of basis of pI, not Mw
Requires very high voltages (5000V)
Requires a long period of time (10h)
Presence of a pH gradient is critical
Degree of resolution determined by slope of pH
gradient and electric field strength
Keeps protein structure intact
Can be scaled up to isolate mg to gms of protein
in a single tube gel run
Column Chromatography
Column Chromatography

Most common (and best) approach to

purifying larger amounts of proteins
Able to achieve the highest level of purity
and largest amount of protein with least
amount of effort and the lowest likelihood of
damage to the protein product
Standard method for pharma industry
Column Chromatography
Can be done either at atmospheric
pressure (gravity feed) or at high
pressure (HPLC, 500-2000 psi)
Four types of chromatography:
Affinity chromatography
Gel filtration (size exclusion)
chromatography
Ion exchange chromatography
Hydrophobic (reverse phase)
chromatography
Affinity Chromatography (AC)

Adsorptive separation in which the

molecule to be purified specifically and
reversibly binds (adsorbs) to a
complementary binding substand (a
ligand) immobilized on an insoluble
support (a matrix or resin)
Purification is 1000X or better from a
single step (highest of all methods)
Preferred method if possible
 AC

Step 1: Attach ligand Step 2: Load protein

to column matrix mixture onto column
 AC

Step 3: Proteins bind

to ligand Step 4: Wash column to remove unwanted
material, elute later
Affinity Chromatography
Used in many applications
Purification of substances from
complex biological mixtures
Separation of native from denatured
forms of proteins
Removal of small amounts of
biomaterial from large amounts of
contaminants
 Affinity Chromatography
The ligand must be readily (and cheaply)
available
Ligand must be attachable (covalently) to
the matrix (typically sepharose) such that it
still retains affinity for protein
Binding must not be too strong or weak
Ideal KD should be between 10-4 & 10-8 M
Elution involves passage of high salt or low
pH buffer after binding
Ligand Specificity
AMP Enzymes with NAD cofactors an ATP
dependent kinases
Arginine Proteases such as prothrombin,
kallikrein, clostripain
Cibacron Blue Serum Albumin, Preablumin
Dye
Heparin Growth factors, cytokines, coagulation
factors
Protein A Fc region of immunoglobulins

Calmodulin Calmodulin regulated kinases, cylcases

and phosphatases
EGTA-copper Proteins with poly-Histidine tails
Size Exclusion Chromatography (SEC)

Molecules are separated according to

differences in their size as they pass
through a hydrophilic polymer
Polymer beads composed of cross-linked
dextran (dextrose) which is highly porous
(like Swiss cheese)
Large proteins come out first (cant fit in
pores), small proteins come out last (get
stuck in the pores)
SEC
Sephadex Structure
Ion Exchange Chromatography
(IEC)
Principle is to separate on basis of
charge adsorption
Positively charged proteins are
reversibly adsorbed to immobilized
negatively charged beads/polymers
Negatively charged proteins are
reversibly adsorbed to immobilized
positively charged beads/polymers
 IEC

Has highest resolving power

Has highest loading capacity
Widespread applicability (almost
universal)
Most frequent chromatographic
technique for protein purification
Used in ~75% of all purifications
IEC Principles
IEC Nomenclature

Matrix is made of porous polymers derivatized

with charged chemicals
Diethylaminoethyl (DEAE) or Quaternary
aminoethyl (QAE) resins are called anion
exchangers because they attract negatively
charged proteins
Carboxymethyl (CM) or Sulphopropyl (SP)
resins are called cation exchangers because
they attract positively charged proteins
IEC Groups
IEC Techniques
Strong ion exchangers (like SP and QAE)
are ionized over a wide pH range
Weak ion exhangers (like DEAE or CM) are
useful over a limited pH range
Choice of resin/matrix depends on:
Scale of separation
Molecular size of components
Isoelectric point of desired protein
pH stability of the protein of interest
Protein pH Stability Curve
+
Attached to
anion exchangers
Net charge on protein

4 5 6 7 8 9 pH

Attached to
cation exchangers
_

Range of pH stability
Polyacrylamide gel electrophoresis and N-terminal amino acid
sequence of D-carnitine dehydrogenase

1. Polyacrylamide gel electrophoresis

A. Native-PAGE B. SDS-PAGE 1. Coomasive brilliant blue R-250 staining
2. Activity staining
3. Marker proteins
4. Purified enzyme

2. N-terminal amino acid sequence of D-carnitine dehydrogenase

5 10 15 20 25 30
M Q N LR R V LI TAAX S G I G R E IAKAF V N E G H L
Estimation of the molecular weight of D-carnitine
dehydrogenase
 1. Membrane
(Module)
2. Pumps
3. Other
Piping
Tanks
Valves
Flowmeter
Manometer

12/4/2017
 REVERSE NANO ULTRA MICRO
FEATURE
OSMOSIS FILTRATION FILTRATION FILTRATION
Symmetrical
Membrane Asymmetrical Asymmetrical Asymmetrical
Asymmetrical
Wall Thickness 150mm 150mm 150-250mm 10-150mm
Film thickness 1mm 1mm 1mm various

Pore size <0.002um <0.002um 0.02-0.2um 0.2-5um

HMWC,
HMWC, mono-
LMWC,Sodiu Macromolecules,
,di-, and aligo-
m, Chloride, proteins, Particulates, clay,
Rejects saccharides,
glucose, polysaccharid bacteria
polyvalent
amino acids, es, viruses
anions
proteins
Tubular, hollow-
Tubular, spiral- Tubular, spiral- Tubular, hollow-
Membrane fibre, spiral-
wound, plate wound, plate fibre, plate &
module wound, plate
& frame & frame frame
& frame
CA, TFC,
Ceramic,
Material CA, TFC CA, TFC CA, TFC, Ceramic
PVDF,
Sintered
Pressure 15-150 bar 5-35 bar 1-10 bar <2 bar
Flux 10-50 (l/m2/hr) 10-100 l/m2/hr 10-200 l/m2/hr 50-1000 l/m2/hr
Some membrane types

Ultrafiltration Microfiltration

RO membrane
MIKROFILTRASI
Menggunakan membran: tipis dan
Tanpa pembentukan cake
microporous
Lubang pori2nya kecil dan sangat
monodisperse
Mempunyai kemampuan menyaring partikel
yang tidak diinginkan
Membran mengikuti hukum Darcys untuk
permeabilitas dan ketahanan yang tinggi thd
aliran. Konvensional ketahanannya rendah.
Perlu dilakukan pembersihan secara berkala
Aliran yang melalui membran lebih rendah
daripada aliran melalui conventional filter
cake. Filter area per liter volume lebih besar
daripada convensional.
Type : Plate and frame, spiral wound and
hollow fiber
Plate and Frame
Spiral Wound
Tubular ceramic
Hollow Fibre
Bacterial Cell Lactose
Casein, whey Minerals
Important terms

Feed or Product
Initial material into system on
feed side of membrane
Retentate or Concentrate
The fraction of the feed which
is rejected by the membrane.
Permeate
The fraction of the feed which
passes through the membrane
 Spiral Wound
Plate and Frame
Tubular
Capilary
Hollow fibre
Pipe

Ceramic
Zeolite
Stainless Steel
 Downstream protein purification
by ultrafiltration concentration and
diafiltration
 MF Applications: late 1990s
Cold sterilization of pharmaceuticals
Cell harvesting
Sterile process filters for gas-phase
Clarification of fruit juices, wine and beer
Ultrapure water in semiconductor industry
Metal recovery (colloidal (hydro)oxides)
Waste water treatment
Separation of oil-water emulsions
Dehydration of lattices
Pretreatment for RO
Eykamp, 1995; Mulder, 1998
 UF Applications: 1980s
Chemical Industry
Electro coat painting recovery
Latex processing
Textile size recovery
Recovery of lubricant oils
Medical Applications
Kidney dialysis
Waste treatment
Recovery of valuable products from
effluents
Cheese whey
Cheryan, 1986
 UF Applications: late 1990s
electro paint recovery, oil-water emulsions
Beverages (juices)
Dairy (milk, whey, cheese making)
Food (gelatin, starch, sugar and proteins)
Textile (sizing, dyes)
Pharmaceutical (enzymes, antibiotics,
pyrogens)
Pulp and paper industry
Leather industry
Water purification
Eykamp, 1995; Mulder, 1998
Factors affecting membrane
structure:
choice of polymer, choice of solvent and
nonsolvent, composition of casting solution,
composition of coagulation bath, temperature
of the casting solution and coagulation bath,
evaporation time, location of the liquid-liquid
demixing gap and crystallization behaviour of
the polymer
 Module Type
Characteristic Flat plate Spiral Shell Hollow Fibre
Wound and
Tube
Packaging Moderate Moderate Low High (9000-30000
density (m2/m3) (200-400) (300-900) (150-300)

Fluid Good Good High Good

management pumping
costs
Suspended Moderate Poor Good Poor
solids
capability
Cleaning Sometimes Sometimes Easy Backflusing possible
difficult difficult
Replacement Sheets or Cartridge Tubes Cartridge
cartridge
DRYING
Reason: The cost of transport can be reduced; the
material is easier to be handle and package; can be
more conveniently stored in the dry state; more
stable than the liquid form.
Instrument: spray dry, in this system the evaporative
cooling protect the enzyme activity.
CRYSTALLIZATION
Is the best way to preserve the enzyme, but the
method for most enzyme still to be developed. The
enzyme should be pure.
 Why immobilized enzymes?

Definition : Immobilization means that the biocatalysts are limited in moving

due to chemically or physically treatment

Reasons
- Reuse of enzyme(reducing cost)
- Easy product separation
- Continuous processing
- Stabilization by immobilization
Limitations
-Cost of carriers and immobilization
-Changes in properties(selectivity)
-Mass transfer limitations
-Activity loss during immobilization
Conventional Immobilization Methods

Adsorption
Covalent
binding

Cross-linking

Immobilization

Entrapment

Encapsulation
Immobilization Adsorption /
Covalent crosslinking Affinity attachment
type Absorption

Identical orientation by
Generally simple Reproducibility and site specific
stability of protein layer immobilization
No manipulation of the Direct immobilization
Advantage protein sample The possibility of by high affinity
controlling the density Easy protein
and environment of the purification and array
immobilized species fabrication
Reproducibility and
stability of protein layer
Some protein denature
The possibility of
and inactive
controlling the density
Unstable binding Non- Non-specific random
and environment of the
specific random and orientation: activity
immobilized species
multi-oriented protein decreased
immobilization: activity Some protein denature
Disadvantage decreased and inactive
Irreproducibility of Additional chemical Difficult application in
results reaction for modification multi-subunit proteins
in vitro The possibility of
elusion of some protein