eKsTraKsI dan PuriFikaSi - WordPress.com

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

Tahun Pengendapan dengan alkohol 1833 (Payen & Persoz) Adsorpsi spesifik amylase dengan substrat insolubel 1910 (Starkenstein) Penggunaan adsorbant untuk pemurnian enzim 1922 (Willstarter) Penggunaan ultrasentrifugasi pertama kali 1923 (Svedberg & Nichols) Kristalisasi Urease

1926 (Summer) 80 enzim terisolasi 1930 Pengenalan resin penukar ion 1935 (Adams & Holmes) Kromatografi adsorpsi 1938-1941 (Zechmeister & Brockman) Metode Pengendapan terfraksi dengan pelarut organik 1946 (Cohn) Kromatografi dengan hidroksi

apatit 1951-1956 (Tiselius & Swingle) Pengenalan penukar ion selulosa 1956 (Sober & Peterson) Pengenalan sepadex dan gel filtrasi 1959 (Porath & Flodin) Elektroforesis Protein 1966 (Vesterberg) Introduksi aktivasi dengan CNBr 1967 (Axen & Porath)

SDS Page elektroforesis 1967 (Shapiro) Induksi konsep kromatografi afinitas 1968 (Cuatrecasas) Kromatografi hidrofob 1971 (Yon, Er El & Shaltiel) Lebih dari 2000 enzim terisolasi 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, 2mercaptoethanol, 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 Enzyme extract Crude Enzyme Dilute Enzyme Conc. Enzyme Treatment I Choosing enzyme source and recovery

Filtration Centrifugation Cell disruption II Removal of whole cells and cell debris from enzyme extract Centrifugation Filtration III Removal of nucleic acids and lipids Precipitation Nucleases Glass wool

IV Concentration and primary purification V Final purification and quality check

Ultrafiltration Precipitation Chromatography Dehydration Gel filtration 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 tidak terlarut Filtrasi 1.0 5 0.2 2.0 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 Gel Filtrasi Ultrasentrifugasi Sentrifugasi zonal Kromatografi penukar ion Mobilitas elektroforesis Gel Elektroforesis Mr

Kromatografi Adsorpsi Elektroforesis Muatan Densitas Enzim Sifat permukaan Stabilitas Kromatografi: Afinitas Hidrofobik Kovalen

Perlakuan : asam-basa suhu Titik isoelektrik Elektro dekantasi Kelarutan Pengendapan Isoelektrik Partisi Cair-cair Pengendapan terfraksi 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 : Aktivitas katalitik Protein

Aktivitas spesifik Kontaminan (enzim & lainnya Aktivitas biologi & Spesifik Elektroforesis Ultrasentrifugasi Gel Filtrasi Pengukuran: Tekanan osmose Pengendapan dengan UF Difusi dan koefisien difusi Gel eksklusi kromatografi Homogenitas Enzim murni

SDS PAGE Mr 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

air Temperatur : shock dingin 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 X100 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 Hemoglobin Pengaturan T T>Kelarutan>, s/d 40-50C Seleksi Protein Protein terdenatura

si Tidak dapat digunakan secara industri D.1.2. PENGENDAPAN DENGAN PELARUT ORGANIK Protein Saling bergabung Enzim Protein 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; dilute solutes independent Usually non linier; dilute solutes interact Nature of operation Steady state

Unsteady; periodic Problems Emulsification; denaturation Solids handling; 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 pH-pKi) Q = Ni/(1 + 10Transcendental equation pKa Values for Ionizable Amno Acids Residue C D E

pKa 10.28 3.65 4.25 Residue H K R pKa 6 10.53 12.43 IEF Principles Increasing pH A

N O D E _ _ _ _ _ _ _ _ _ + + + + +

+ + + + pI = 5.1 pI = 6.4 pI = 8.6 C A T H O D E 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 to column matrix Step 2: Load protein 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 Net charge on protein + Attached to anion exchangers

4 5 6 7 8 Attached to cation exchangers _ Range of pH stability 9 pH Polyacrylamide gel electrophoresis and Nterminal amino acid sequence of D-carnitine dehydrogenase

1. Polyacrylamide gel electrophoresis A. Native-PAGE B. SDS-PAGE 1. 2. 3. 4. Coomasive brilliant blue R-250 staining Activity staining Marker proteins Purified enzyme 2. N-terminal amino acid sequence of D-carnitine dehydrogenase 5 10 15 20

25 30 MQNLRRVLITAAXSGIGREIAKAFVNEGHL Estimation of the molecular weight of D-carnitine dehydrogenase 1. Membrane (Module) 2. Pumps 3. Other Piping Tanks Valves Flowmeter Manometer 02/28/20

FEATURE REVERSE OSMOSIS NANO FILTRATION ULTRA FILTRATION Membrane Asymmetrical Asymmetrical Asymmetrical Wall Thickness

150m 150m 150-250m 10-150m Film thickness 1m 1m 1m various Pore size

<0.002um <0.002um 0.02-0.2um 0.2-5um HMWC, LMWC,Sodiu m, Chloride, glucose, amino acids, proteins HMWC, mono-,di-, and aligosaccharides, polyvalent

anions Macromolecules, proteins, polysaccharid es, viruses Particulates, clay, bacteria Tubular, hollowfibre, spiralwound, plate & frame Tubular, hollowfibre, plate & frame CA, TFC, Ceramic, PVDF, Sintered

Rejects Membrane module Tubular, spiralwound, plate & frame Tubular, spiralwound, plate & frame MICRO FILTRATION Symmetrical Asymmetrical Material CA, TFC

CA, TFC CA, TFC, Ceramic 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 RO membrane Microfiltration MIKROFILTRASI Tanpa pembentukan cake Menggunakan membran: tipis dan 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 Casein, whey Lactose 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 Wound Shell and Tube

Hollow Fibre Packaging density (m2/m3) Moderate (200-400) Moderate (300-900) Low High (9000-30000 (150-300) Fluid management Good

Good High pumping costs Good Suspended solids capability Moderate Poor Good Poor

Cleaning Sometimes difficult Sometimes difficult Easy Backflusing possible Replacement Sheets or cartridge Cartridge

Tubes 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 Limitations - Reuse of enzyme(reducing cost) -Cost of carriers and immobilization - Easy product separation -Changes in properties(selectivity) - Continuous processing -Mass transfer limitations - Stabilization by immobilization

-Activity loss during immobilization Conventional Immobilization Methods Adsorption Covalent binding Cross-linking Immobilization Entrapment Encapsulation Immobilization type Adsorption /

Absorption Generally simple Advantage Disadvantage No manipulation of the protein sample Some protein denature and inactive Unstable binding Nonspecific random and multi-oriented protein immobilization: activity decreased Irreproducibility of results

Covalent crosslinking Reproducibility and stability of protein layer The possibility of controlling the density and environment of the immobilized species Non-specific random orientation: activity decreased Some protein denature and inactive Additional chemical reaction for modification in vitro Affinity attachment

Identical orientation by site specific immobilization Direct immobilization by high affinity Easy protein purification and array fabrication Reproducibility and stability of protein layer The possibility of controlling the density and environment of the immobilized species Difficult application in multi-subunit proteins The possibility of elusion of some protein

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