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EKsTraKsI dan PuriFikaSi. A. Pendahuluan Beberapa teknik isolasi enzim secara umum dikelompokan menjadi: –Metode klasik berupa distilasi dan ekstraksi.

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Presentasi berjudul: "EKsTraKsI dan PuriFikaSi. A. Pendahuluan Beberapa teknik isolasi enzim secara umum dikelompokan menjadi: –Metode klasik berupa distilasi dan ekstraksi."— Transcript presentasi:

1 eKsTraKsI dan PuriFikaSi

2 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

3 Penemuan separasi enzim PenemuanTahun Pengendapan dengan alkohol1833 (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 Urease1926 (Summer) 80 enzim terisolasi1930 Pengenalan resin penukar ion1935 (Adams & Holmes)

4 Kromatografi adsorpsi (Zechmeister & Brockman) Metode Pengendapan terfraksi dengan pelarut organik 1946 (Cohn) Kromatografi dengan hidroksi apatit (Tiselius & Swingle) Pengenalan penukar ion selulosa1956 (Sober & Peterson) Pengenalan sepadex dan gel filtrasi 1959 (Porath & Flodin) Elektroforesis Protein1966 (Vesterberg) Introduksi aktivasi dengan CNBr1967 (Axen & Porath) SDS Page elektroforesis1967 (Shapiro) Induksi konsep kromatografi afinitas 1968 (Cuatrecasas) Kromatografi hidrofob1971 (Yon, Er El & Shaltiel) Lebih dari 2000 enzim terisolasi1980

5 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

6 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.

7 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

8 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

9 Skema umum isolasi dan purifikasi enzim Materi Primer AnimalAnimal VegetalVegetal MikrobaMikroba Ekstrak total Ekstrak kasar Enzim murni Tahap Ekstraksi Tahap Fraksionasi Tahap Purifikasi

10 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 BacteriaBacillus subtilis B. Amyloliquifaciens L. spesies FungiAspergillus sp. Penicillium sp. Mucor Rhizobium S. cerevesiae

11 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 EnzymePlant source Ascorbate oxidaseCurcubita species UreaseJack bean BromealinPineapple stem/fruit AmylaseBarley Pectine esteraseCitrus fruits PhytaseWheat,rye, triticale

12 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 esteraseBovine erythrocytes ArginaseBeef liver Creatine kinaseRabbit muscle, beef heart Aldose reductaseBeef eyes UricasePorcine liver TrypsinMammalian pancreas

13 STEP IN ENZYME PURIFICATION StepTargetTreatment IChoosing enzyme source and recovery Filtration Centrifugation Cell disruption IIRemoval of whole cells and cell debris from enzyme extract Centrifugation Filtration IIIRemoval of nucleic acids and lipids Precipitation Nucleases Glass wool IVConcentration and primary purification Ultrafiltration Precipitation Chromatography Dehydration VFinal purification and quality check Gel filtration Ion exchange Affinity Hydrophobic interaction Chromatofocussing HPLC Enzyme extract Crude Enzyme Dilute Enzyme Conc. Enzyme

14 Profil Proses TingkatanProduk ProsesKons. (g/l)Kwalitas (%) PemanenanFermentasi0.1 – 50.1 – 1.0 Pengambilan bahan tidak terlarut Filtrasi1.0 – 50.2 – 2.0 IsolasiEkstraksi5 – 501 – 10 PurifikasiKromatografi50 – – 80 Produk akhirKristalisasi50 – – 100

15 Teknik separasi dan purifikasi berdasarkan sifatnya EnzimEnzim Mr Densitas Sifat permukaan Stabilitas Muatan Titik isoelektrik Kelarutan Gel Elektroforesis Ultrasentrifugasi Sentrifugasi zonal Kromatografi Adsorpsi Kromatografi: Afinitas Hidrofobik Kovalen Perlakuan : asam-basa suhu Partisi Cair-cair Pengendapan terfraksi dengan garam atau pelarut organik Pengendapan Isoelektrik Elektro dekantasi Mobilitas elektroforesis Elektroforesis Kromatografi penukar ion Ultrasentrifugasi Dialisa Gel Filtrasi

16 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

17 Prosedur umum kontrol kwalitas enzim murni Enzim murni Aktivitas biologi & Spesifik Aktivitas biologi & Spesifik Pengukuran : Aktivitas katalitik Protein Aktivitas spesifik Kontaminan (enzim & lainnya Homogenitas Mr Polimorfisme, Mr Komposisi & Sequence Elektroforesis Ultrasentrifugasi Gel Filtrasi Pengukuran: Tekanan osmose Pengendapan dengan UF Difusi dan koefisien difusi Gel eksklusi kromatografi SDS PAGE Metode Sanger Kemasan Stabilitas Label Penggunaan

18 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)

19 Metode ekstraksi MetodePerlakuan 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 spesialPelarut organik: butanol, aseton dan pelarut lipid Pembekuan dan pencairan Penggunaan deterjenNa-deoksi kolat, Tween 20, Teepol XL, Triton X- 100 Ekstraksi dengan enzimOtolisis: proteolisa dan lipolisa Penggunaan enzim pemurni (tripsin, lipase)

20 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

21 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

22 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

23 D.1.2. PENGENDAPAN PADA ISOELEKTRIK D.1.3. EFEK TEMPERATUR pIpI Pengaturan pH larutan Kelarutan minimum, mengendap Pengaturan TSeleksi Protein T>Kelarutan>, s/d 40-50C Protein terdenaturasi Tidak dapat digunakan secara industri Protein Globular Hemoglobin Hemoglobin

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

25 VariableExtractionAdsorption CapacityHighLow SelectivityModerateHigh Nature of equilibriumOften linier; dilute solutes independent Usually non linier; dilute solutes interact Nature of operationSteady stateUnsteady; periodic ProblemsEmulsification; denaturation Solids handling; compressible packing

26 Electrophoresis

27 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

28 Isoelectric Focusing (IEF)

29 Isoelectric Point (pI) The pH at which a protein has a net charge=0 Q =  Ni/( pH-pKi ) Transcendental equation

30 IEF Principles ANODEANODE __________________ CATHODECATHODE Increasing pH pI = 8.6 pI = 6.4 pI = 5.1

31 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

32 Column Chromatography

33 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

34 Column Chromatography Can be done either at atmospheric pressure (gravity feed) or at high pressure (HPLC, psi) Four types of chromatography: –Affinity chromatography –Gel filtration (size exclusion) chromatography –Ion exchange chromatography –Hydrophobic (reverse phase) chromatography

35 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

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

37 A C Step 3: Proteins bind to ligand Step 4: Wash column to remove unwanted material, elute later

38 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

39 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 K D should be between & M Elution involves passage of high salt or low pH buffer after binding

40 LigandSpecificity AMPEnzymes with NAD cofactors an ATP dependent kinases ArginineProteases such as prothrombin, kallikrein, clostripain Cibacron Blue Dye Serum Albumin, Preablumin HeparinGrowth factors, cytokines, coagulation factors Protein AFc region of immunoglobulins CalmodulinCalmodulin regulated kinases, cylcases and phosphatases EGTA-copperProteins with poly-Histidine tails

41 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)

42 SEC

43 Sephadex Structure

44 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

45 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

46 IEC Principles

47 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

48 IEC Groups

49 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

50 Protein pH Stability Curve + _ Net charge on protein pH Attached to cation exchangers Attached to anion exchangers Range of pH stability

51

52

53 Polyacrylamide gel electrophoresis and N-terminal amino acid sequence of D-carnitine dehydrogenase B. SDS-PAGE B. SDS-PAGE A. Native-PAGE 1. Polyacrylamide gel electrophoresis 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 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

54 Estimation of the molecular weight of D-carnitine dehydrogenase

55 1. Membrane (Module) 2. Pumps 3. Other Piping Tanks Valves Flowmeter Manometer 6/8/2015

56 FEATURE REVERSE OSMOSIS NANO FILTRATION ULTRA FILTRATION MICRO FILTRATION MembraneAsymmetrical Symmetrical Asymmetrical Wall Thickness 150  m  m  m Film thickness 1m1m1m1m1m1m various Pore size<0.002um um0.2-5um Rejects HMWC, LMWC,Sodiu m, Chloride, glucose, amino acids, proteins HMWC, mono-,di-, and aligo- saccharides, polyvalent anions Macromolecules, proteins, polysaccharid es, viruses Particulates, clay, bacteria Membrane module Tubular, spiral- wound, plate & frame Tubular, hollow- fibre, spiral- wound, plate & frame Tubular, hollow- fibre, plate & frame MaterialCA, TFC CA, TFC, Ceramic CA, TFC, Ceramic, PVDF, Sintered Pressure bar5-35 bar1-10 bar<2 bar Flux10-50 (l/m 2 /hr) l/m 2 /hr l/m 2 /hr l/m 2 /hr

57 Some membrane types RO membrane Ultrafiltration Microfiltration

58 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 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

59 Plate and Frame

60 Spiral Wound

61 Tubular ceramic

62 Hollow Fibre

63 Bacterial Cell Casein, whey Minerals Lactose

64 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

65

66

67 Spiral Wound Plate and Frame Tubular Capilary Hollow fibre Pipe Ceramic Zeolite Stainless Steel

68 Downstream protein purification by ultrafiltration concentration and diafiltration

69 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

70 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

71 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

72 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

73 Characteristic Module Type Flat plateSpiral Wound Shell and Tube Hollow Fibre Packaging density (m 2 /m 3 ) Moderate ( ) Moderate ( ) Low ( ) High ( Fluid management Good High pumping costs Good Suspended solids capability ModeratePoorGoodPoor CleaningSometimes difficult EasyBackflusing possible ReplacementSheets or cartridge CartridgeTubesCartridge

74 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.

75 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

76 Immobilization Cross-linking Adsorption Covalent binding Entrapment Encapsulation Conventional Immobilization Methods

77 Immobilization type Adsorption / Absorption Covalent crosslinkingAffinity attachment Advantage Disadvantage  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  Difficult application in multi-subunit proteins  The possibility of elusion of some protein


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