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Kelompok 10RadityaRainer CYongkiRepublikBioseparasi Papain sebagai bahan baku obatIsolasi PapainGetah pepaya dikumpulkan dengan konvensional digunakan sebagai bahan baku utama papainBahan Pohon pepaya lokalStandard papain, polyacrylamide, bis-acrylamide, ammonium persulfate and PEG 6000 SigmaAldrich (St. Louis, USA),Casien (Hammarsten) BDH (Poole, Eng-land).Isolasi GetahLatex atau getah diisolasi dari pohon pepayaGetah dikumpulkan dengan perangkat yang terpasang pada batang pohonGetah disimpan dalam botol plastik pada suhu -200CSegar lateks dikumpulkan dari C. pepaya lokal berkembang. Awalnya, empat sampai enam Insisi longitudinal dilakukan pada buah mentah menggunakan stainless steel pisau. The memancarkan latexwas diperbolehkan untuk berlari menuruni buah dan menetes ke dalam pengumpulan perangkat yang terpasang di sekitar batang. Setelah koleksi, yang latexwas ditransfer untuk botol plastik dan disimpan pada -20 C. 4Purifikasi PapainSalt precipitation (pengendapan garam) 2 tahap

Aqueous two-phase system (sistem dua fase)Salt precipitation 1Getah dicampur dengan cystein dengan perbandingan 3:1 (w/v) disuspensi dengan 6M HCl samapi pH 5.6 dan diaduk dengan stirer 15 menit pada 40CHasil larutan ditambahkan 6M NaOH sampai pH 9Zat yang tidak larut dihilangkan dengan sentrifugasi 9000 g 30 menit, 40CSupernatant diambil ditentukan kandungan protein

Purication of papain from latex by two-step saltPrecipitationThawed latexwasmixedwith 40mMcysteine at a ratio of 3:1 (w/v) and the suspension was adjusted to pH 5.6 using6M HCl and then stirred for 15min at 4 C. The mixture was ltered and pHof the ltrate was adjusted to 9.0 using 6M NaOH. The insoluble material wasremoved by centrifugation at 9000g for 30min at 4 C. The protein content inthe supernatantwas determined and then adjustedwithwater before precipitationwith (NH4)2SO4 at 45%saturation. The salt-enriched solutionwas slowly stirredat 4 C for 30min. The precipitate was collected by centrifugation as above, anddissolved using 20mM cysteine. The solution was kept at 4 C before addingsodium chloride (10%, w/v). The mixture was slowly stirred for 30min beforeseparating the precipitated papain by centrifugation. The enzyme was dissolvedin water and dialyzed overnight at 4 C against three changes (1 l each) of water.The dialysate was nally lyophilized (in a freeze-dryer, LY5FM-ULE, SnijdersScientic BV, Tilburg, The Netherlands) to obtain puried papain powder.6Salt precipitation 2Supernatan dinetralkan dengan penambahan airPengendapan dengan menambahkan (NH4)2SO4 kemudian diaduk dengan stirrer pada suhu 40C selama 30 menitSentrifugasi 9000 g 30 menit, 40CPesipitat diambil ditambahkan 20mM cysteinLarutan dijaga 40C kemudian ditambahkan 10%NaOH (w/v) distirrer selama 30 menit Sentrifugasi untuk memisahkan padatan papainPadatan dilarutkan dalam air dan didialisis pada 40C dengan tiga kali pergantian air (1 L)Endapan yang didapatkan berupa enzim papain, di lyophilized (freeze dryer) maka didapatkan powder papain

The protein content inthe supernatantwas determined and then adjustedwithwater before precipitationwith (NH4)2SO4 at 45%saturation. The salt-enriched solutionwas slowly stirredat 4 C for 30min. The precipitate was collected by centrifugation as above, anddissolved using 20mM cysteine. The solution was kept at 4 C before addingsodium chloride (10%, w/v). The mixture was slowly stirred for 30min beforeseparating the precipitated papain by centrifugation. The enzyme was dissolvedin water and dialyzed overnight at 4 C against three changes (1 l each) of water.The dialysate was nally lyophilized (in a freeze-dryer, LY5FM-ULE, SnijdersScientic BV, Tilburg, The Netherlands) to obtain puried papain powder.

7Salt precipitation 8Extraction in aqueous two-phase systemGetah dicampur dengan air dan pH diatur dengan 6M HCl.Ekstraksi dua fase diperlukan penambahan PEG (Polietilen Glikol) dan (NH4)2SO4 pada getah ( 30 gr getah)Hasil pencampuran ditambahkan air sampai dengan 50 gr, pH diatur dengan menambahakan 6M HCl atau 6 M NaOHLarutan diaduk selama 15 menit dengan perlahan.Kedua fase yang terbentuk dipisahkan dengan sentrifugasi 9000 g selama 30 menit pada 40CAliquot diambil untuk dianalisis protein dan aktifitas protease.Keberadaan papain dalam fraksi ditentukan dengan analisis gel elektrophoresis katode dan FPLCThe thawed latex was mixed with water and pH adjusted using 6M HCl.For extraction in aqueous two-phase system, dened amounts of solid PEG and(NH4)2SO4 were added to the latex preparation (30 g), and the totalmixture wasmade up to 50 g with water. When studying the effect of pH on protease/papainpartitioning, the latexwas adjusted to the desired pHusing 6MHCl or 6MNaOHsolutions prior to mixing with the phase components. The mixture was thengently shaken for 15min. The two phases were separated by centrifugation at9000g for 30min at 4 C. Aliquots of the phases were taken for determinationof protein concentration and protease activity. For calculation of protease activityin the two phases, volumes of the respective phaseswere taken into consideration.The presence of papain was veried by cathodic gel electrophoresis and FPLC.9Extraction in aqueous two-phase systemAnalisis dengan FPLC, fraksi atas sebanyak 6 ml dari larutan didialisis tiga kali pada 40C dengan 50 mM sodium asetat buffer pH 5PEG dipisahakan dari larutan dengan Chromatografi ion-exchangeLarutan dimasukan dalam kolom CM-selulosa (1.5 cm x 2cm) diequiliberasi dengan 50 mM asetat buffer pH 5Enzim dielusi dari kolom dengan buffer NaCl 1 MPapain sebagaian besar terelusi didepan kedua bufferFraksi ini dikumpulkan, didialisis dan lyophilized (freeze dryer) didapatkan powder papainFor analysis of papain purity by FPLC, the top phase (6ml) from an aqueouswo-phase systemwas dialyzed three times at 4 Cagainst 50mMsodiumacetatebuffer at pH 5. The dialyzed solution was subjected to ion-exchange chromatog-aphy for separation of the enzyme from PEG [20]. The solution was loaded ona CM-cellulose column (1.5 cm2 cm) equilibrated with 50mMacetate buffer,pH 5 and after washing the enzyme was eluted from the column with the buffercontaining 1M NaCl. Papain was largely eluted at the front of the two buffers.The fractions containing protease activity were pooled, dialyzed and lyophilizedo obtain puried papain powder.10Aqueous two-phase systemAnalisis kemurnian dengan Fast Protein Liquid Chromatography25 l larutan papain dalam 20 mM glycine-NaOH buffer digunakan di dalam kolom Mono Q HR 5/5 (1 ml) yang di cuci dengan 5 ml buffer yang samaPapain dan protein lainnya terelusi oleh gradien linear sodium klorida dari 0 0,5 M (total volume 24 ml) dengan laju 1,0 ml/minFraksi 1 ml di ambil dan diplot kromatografi A280 dan komposisi gradien vs volume elusi direcord Peak elusi dari papain ditentukan menggunakan papain standarPersentase luas peak papain didapatkan dari automatic integratorKemurnian papain diketahui dari persentase luas peak papain berbanding luas peak totalAnalisis aktifitas enzim papain100 l larutan enzim dipreparasi dahulu dengan dicampurkan 200 l 50 mM cysteine-20 mM EDTA dan 700 l 50 mM Tris-HCl buffer pada pH 8Campuran tersebut diinkubasi selama 5 menit pada suhu 37C direaksikan dengan 1 ml 1% (w/v) larutan caseinSetelah 10 menit, reaksi dihentikan dengan menambahkan 3 ml 5% (v/v) trichloroacetic acid (TCA) pada larutan tersebut dan didinginkan selama 1 jamCampuran itu kemudian disentrifugasi dan diambil supernatannya untuk diukur absorbansinya pada 275 nmSatuan unit aktifitas protease tersebut diukur berdasarkan jumlah enzim yang memberikan produk digesti terlarut yang menghasilkan kenaikan absorbansi pada 275 nm setara dengan 1 mol dari tyrosine/min pada kondisi percobaanPapain assayGel Elektrophoresis katode-polyacryamide

Standar PapainCrude LatexSalt precipitate4. Fraksi atas 2 phase aqueous5. Fraksi bawah Bottom Fig. 1. Cathodic polyacrylamide gel electrophoresis of papain during purica-tion by two-step salt precipitation and extraction in aqueous two-phase systemconsisting of 8% PEG15% (NH4)2SO4. The samples in the different lanesrepresent: standard papain (lane 1, 6 g), crude latex extract (lane 2), puriedpapain from a two-step salt precipitation (lane 3), and from top phase of the sys-tem consisting of 8% (w/w) PEG15% (w/w) (NH4)2SO4 (lane 4), and bottomphase of the same two-phase system (lane 5) after partitioning of latex.14Salt precipitation

Aqueous two-phase system

Hasil Papain Papain murni didapatkan pada fase atas FPLC dengan 1/4 volume dari keseluruhanOptimal precipitation dan two phase sistem purity (100% versus 89%) dari papaya latex.

(top phase volume=6ml bottom phase volume = 25.5 ml).17FPLC

Fast protein liquid chromatography (FPLC), is a form of liquid chromatography similar to high-performance liquid chromatography[citation needed] that is used to separate or purify proteins and other polymers from complex mixtures. FPLC system is a complete system for laboratory scale chromatographic separations of proteins and other biomolecules.The columns used in FPLC are large [mm id] tubes that contain small [] particles or gel beads that are known as stationary phase. The chromatographic bed is composed by the gel beads inside the column and the sample is introduced into the injector and carried into the column by the flowing solvent. As a result of different components adhering to or diffusing through the gel, the sample mixture gets separated.[3] Columns used with an FPLC can separate macromolecules based on size, charge distribution (ion exchange), hydrophobicity, reverse-phase or biorecognition (as with affinity chromatography).[4] For easy use, a wide range of pre-packed columns for techniques such as ion exchange, gel filtration (size exclusion), hydrophobic interaction, and affinity chromatography are available.[5] FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum pressure of 3-4 MPa (435-580 psi). Thus, if the pressure of HPLC can be limited, each FPLC column may also be used in an HPLC machine.18Estimasi Kasar Ekonomi AnalisisHarga peralatan AnalisisFPLC : $3500Gel Elektrophoresis katode-polyacryamide : $2500Harga papain 37.5 euro/ 225 gr

Ekonomi dianalisis lebih lanjut. Coba dicari untuk lebih ada harga tiap alat dan bahannya lagi.19Ekonomi AnalisisPapain Enzyme Dietary Supplement Concentrate The product is standardized 400 MCU/ gram Bulk Food Grade USP Powder Packaging: 225 grams Price $37.25 ea.20Spesifikasi Papain Papain adalah cysteine protease daripeptidase (C1 family).Papain terdiri atas sebuah rantai polypeptide dengan tiga ikatan disulfida dan a sulfhydryl group sebagai pengaktif enzyme. Spectral properties:max: 278 nm Extinction coefficient, E1% = 25 Extinction coefficient, EmM = 57.6 (at 280 nm) Definisi Unit: 1 unit menghidrolisis 1.0 mole of N--benzoyl-L-arginine ethyl ester (BAEE) per menit pada pH 6.2 suhu 25 C. Berat Molekul : 23,406 Da (amino acid sequence)Optimal pH for activity : 6.0-7.0 Temperature Optimum : 65 C

Sigma Alderich