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 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: Hal yang perlu diperhatikan 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 (4ºc) 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 Target Treatment I Choosing enzyme source and recovery Filtration Centrifugation Cell disruption II Removal of whole cells and cell debris from enzyme extract III Removal of nucleic acids and lipids Precipitation Nucleases Glass wool IV Concentration and primary purification Ultrafiltration Chromatography Dehydration V Final purification and quality check Gel filtration Ion exchange Affinity Hydrophobic interaction Chromatofocussing HPLC Enzyme extract Crude Enzyme Dilute Enzyme Conc. Enzyme

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 90 – 100

Teknik separasi dan purifikasi berdasarkan sifatnya Ultrasentrifugasi Dialisa Gel Filtrasi Kromatografi penukar ion Elektroforesis Ultrasentrifugasi Sentrifugasi zonal Mobilitas elektroforesis Gel Elektroforesis Mr Muatan Elektro dekantasi Enzim Densitas Titik isoelektrik Kelarutan Sifat permukaan Stabilitas Pengendapan Isoelektrik Kromatografi Adsorpsi Perlakuan : asam-basa suhu Kromatografi: Afinitas Hidrofobik Kovalen 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 Elektroforesis Ultrasentrifugasi Gel Filtrasi Pengukuran: Tekanan osmose Pengendapan dengan UF 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 sel Mekanik : potong, pecah dan homogenasi 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 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 D.1.3. EFEK TEMPERATUR pI Kelarutan minimum, mengendap Pengaturan pH larutan Protein Globular Hemoglobin T>Kelarutan>, s/d 40-50C Pengaturan T Seleksi Protein Protein terdenaturasi Tidak dapat digunakan secara industri

D.1.2. PENGENDAPAN DENGAN PELARUT ORGANIK Alkohol Isopropanol (paling banyak digunakan) untuk enzim ekstraseluler; amiloglukosidase Methanol Aseton dan etil eter: protein sedikit larut maka perlu jumlah yang banyak Enzim Protein Saling bergabung Protein mengendap <: konstanta dielektrikum & kestabilan Penambahan pelarut pada T< shg tidak mendenaturasi Tambah pelarut organik

COMPARISON BETWEEN ADSORPTION AND EXTRACTION Variable Extraction Adsorption Capacity High Low Selectivity Moderate 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 Q = S Ni/(1 + 10pH-pKi) Transcendental equation

IEF Principles A N O D E + _ C T H Increasing pH pI = 8.6 pI = 6.4

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

A C Step 1: Attach ligand Step 2: Load protein to column matrix mixture onto column

A C 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 Dye Serum Albumin, Preablumin 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 (can’t 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

I E C 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 A. Native-PAGE B. SDS-PAGE 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 M Q N L R R V L I T A A X S G I G R E I A K A F V N E G H L

Estimation of the molecular weight of D-carnitine dehydrogenase

Membrane System 1. Membrane (Module) 2. Pumps 3. Other Piping Tanks Valves Flowmeter Manometer 4/16/2017

Symmetrical Asymmetrical FEATURE REVERSE OSMOSIS NANO FILTRATION ULTRA MICRO Membrane Asymmetrical Symmetrical Asymmetrical Wall Thickness 150mm 150-250mm 10-150mm Film thickness 1mm various Pore size <0.002um 0.02-0.2um 0.2-5um Rejects HMWC, LMWC,Sodium, Chloride, glucose, amino acids, proteins HMWC, mono-,di-, and aligo-saccharides, polyvalent anions Macromolecules, proteins, polysaccharides, viruses Particulates, clay, bacteria Membrane module Tubular, spiral-wound, plate & frame Tubular, hollow-fibre, spiral-wound, plate & frame Tubular, hollow-fibre, plate & frame Material CA, TFC CA, TFC, Ceramic 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 microporous Lubang pori2nya kecil dan sangat monodisperse Mempunyai kemampuan menyaring partikel yang tidak diinginkan Membran mengikuti hukum Darcy’s 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 Tanpa pembentukan cake

Plate and Frame

Spiral Wound

Tubular ceramic

Hollow Fibre

Bacterial Cell Lactose Casein, whey Minerals

Important terms Feed or Product Retentate or Concentrate Permeate 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

Comparison: Depth Filter and Membrane Filter

Basic principle

Membrane Material and Configuration Polymeric Inorganic Spiral Wound Plate and Frame Tubular Capilary Hollow fibre Pipe Polymeric Ceramic Zeolite Stainless Steel Inorganic

Amersham Biosciences Downstream protein purification by ultrafiltration concentration and diafiltration Amersham Biosciences

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 Medical Applications 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 (150-300) High (9000-30000 Fluid management Good High pumping costs Suspended solids capability Moderate Poor Cleaning Sometimes difficult Easy Backflusing possible Replacement Sheets or cartridge Cartridge Tubes

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 type Adsorption / Absorption Covalent crosslinking Affinity attachment Advantage Generally simple  No manipulation of the protein sample  Reproducibility and stability of protein layer  The possibility of controlling the density and environment of the immobilized species  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  Some protein denature and inactive  Unstable binding Non-specific random and multi-oriented protein immobilization: activity decreased  Irreproducibility of results  Non-specific random orientation: activity decreased  Some protein denature and inactive  Additional chemical reaction for modification in vitro Disadvantage  Difficult application in multi-subunit proteins  The possibility of elusion of some protein