Bioseparasi Papain sebagai bahan baku obat

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1 Bioseparasi Papain sebagai bahan baku obat
Kelompok 10 Raditya Rainer C Yongki Republik

2 Isolasi Papain Getah pepaya dikumpulkan dengan konvensional digunakan sebagai bahan baku utama papain

3 Bahan Pohon pepaya lokal
Standard papain, polyacrylamide, bis-acrylamide, ammonium persulfate and PEG 6000 Sigma–Aldrich (St. Louis, USA), Casien (Hammarsten) BDH (Poole, Eng-land).

4 Isolasi Getah Latex atau getah diisolasi dari pohon pepaya
Getah dikumpulkan dengan perangkat yang terpasang pada batang pohon Getah disimpan dalam botol plastik pada suhu -200C Segar 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.

5 Purifikasi Papain Salt precipitation (pengendapan garam) 2 tahap
Aqueous two-phase system (sistem dua fase)

6 Salt precipitation 1 Getah dicampur dengan cystein dengan perbandingan 3:1 (w/v) disuspensi dengan 6M HCl samapi pH 5.6 dan diaduk dengan stirer 15 menit pada 40C Hasil larutan ditambahkan 6M NaOH sampai pH 9 Zat yang tidak larut dihilangkan dengan sentrifugasi 9000 g 30 menit, 40C Supernatant diambil ditentukan kandungan protein Purification of papain from latex by two-step salt Precipitation Thawed latexwasmixedwith 40mM cysteine at a ratio of 3:1 (w/v) and the suspension was adjusted to pH 5.6 using 6M HCl and then stirred for 15min at 4 ◦C. The mixture was filtered and pH of the filtrate was adjusted to 9.0 using 6M NaOH. The insoluble material was removed by centrifugation at 9000×g for 30min at 4 ◦C. The protein content in the supernatantwas determined and then adjustedwithwater before precipitation with (NH4)2SO4 at 45%saturation. The salt-enriched solutionwas slowly stirred at 4 ◦C for 30min. The precipitate was collected by centrifugation as above, and dissolved using 20mM cysteine. The solution was kept at 4 ◦C before adding sodium chloride (10%, w/v). The mixture was slowly stirred for 30min before separating the precipitated papain by centrifugation. The enzyme was dissolved in water and dialyzed overnight at 4 ◦C against three changes (1 l each) of water. The dialysate was finally lyophilized (in a freeze-dryer, LY5FM-ULE, Snijders Scientific BV, Tilburg, The Netherlands) to obtain purified papain powder.

7 Salt precipitation 2 Supernatan dinetralkan dengan penambahan air
Pengendapan dengan menambahkan (NH4)2SO4 kemudian diaduk dengan stirrer pada suhu 40C selama 30 menit Sentrifugasi 9000 g 30 menit, 40C Pesipitat diambil ditambahkan 20mM cystein Larutan dijaga 40C kemudian ditambahkan 10%NaOH (w/v) distirrer selama 30 menit Sentrifugasi untuk memisahkan padatan papain Padatan 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 in the supernatantwas determined and then adjustedwithwater before precipitation with (NH4)2SO4 at 45%saturation. The salt-enriched solutionwas slowly stirred at 4 ◦C for 30min. The precipitate was collected by centrifugation as above, and dissolved using 20mM cysteine. The solution was kept at 4 ◦C before adding sodium chloride (10%, w/v). The mixture was slowly stirred for 30min before separating the precipitated papain by centrifugation. The enzyme was dissolved in water and dialyzed overnight at 4 ◦C against three changes (1 l each) of water. The dialysate was finally lyophilized (in a freeze-dryer, LY5FM-ULE, Snijders Scientific BV, Tilburg, The Netherlands) to obtain purified papain powder.

8 Salt precipitation Pelarutan dengan cystein Pengaturan pH (6M HCl)
Penambahan (6 M NaOH) Sentrifugasi (Supernatan) Penambahan air Pengendapan dengan (NH4)2SO4 Sentrifugasi (Presipitat) Penambahan cyctein Penambahan NaOH (sentrifugasi) Dialisis dengan air Freeze dry (Powdwer papain)

9 Extraction in aqueous two-phase system
Getah 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 NaOH Larutan diaduk selama 15 menit dengan perlahan. Kedua fase yang terbentuk dipisahkan dengan sentrifugasi 9000 g selama 30 menit pada 40C Aliquot diambil untuk dianalisis protein dan aktifitas protease. Keberadaan papain dalam fraksi ditentukan dengan analisis gel elektrophoresis katode dan FPLC The thawed latex was mixed with water and pH adjusted using 6M HCl. For extraction in aqueous two-phase system, defined amounts of solid PEG and (NH4)2SO4 were added to the latex preparation (30 g), and the totalmixture was made up to 50 g with water. When studying the effect of pH on protease/papain partitioning, the latexwas adjusted to the desired pHusing 6MHCl or 6MNaOH solutions prior to mixing with the phase components. The mixture was then gently shaken for 15min. The two phases were separated by centrifugation at 9000×g for 30min at 4 ◦C. Aliquots of the phases were taken for determination of protein concentration and protease activity. For calculation of protease activity in the two phases, volumes of the respective phaseswere taken into consideration. The presence of papain was verified by cathodic gel electrophoresis and FPLC.

10 Extraction in aqueous two-phase system
Analisis dengan FPLC, fraksi atas sebanyak 6 ml dari larutan didialisis tiga kali pada 40C dengan 50 mM sodium asetat buffer pH 5 PEG dipisahakan dari larutan dengan Chromatografi ion- exchange Larutan dimasukan dalam kolom CM-selulosa (1.5 cm x 2cm) diequiliberasi dengan 50 mM asetat buffer pH 5 Enzim dielusi dari kolom dengan buffer NaCl 1 M Papain sebagaian besar terelusi didepan kedua buffer Fraksi ini dikumpulkan, didialisis dan lyophilized (freeze dryer) didapatkan powder papain For analysis of papain purity by FPLC, the top phase (6ml) from an aqueous wo-phase systemwas dialyzed three times at 4 ◦Cagainst 50mMsodiumacetate buffer 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 on a CM-cellulose column (1.5 cm×2 cm) equilibrated with 50mMacetate buffer, pH 5 and after washing the enzyme was eluted from the column with the buffer containing 1M NaCl. Papain was largely eluted at the front of the two buffers. The fractions containing protease activity were pooled, dialyzed and lyophilized o obtain purified papain powder.

11 Aqueous two-phase system
Penambahan air dan Pengaturan pH (6M HCl) Penambahan PEG (Polietilen Glikol) dan (NH4)2SO4 Sentrifugasi (fraksi) Aliquot dianalisis Fraksi atas didialisis sodium asetat PEG dipisahkan kromatografi ion exchange Kolom CM-Selulosa Asetat buffer NaCl Buffer Fraksi dikumpulkan Freeze dryer (powder papain)

12 Analisis kemurnian dengan Fast Protein Liquid Chromatography
25 µl 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 sama Papain dan protein lainnya terelusi oleh gradien linear sodium klorida dari 0 – 0,5 M (total volume 24 ml) dengan laju 1,0 ml/min Fraksi 1 ml di ambil dan diplot kromatografi A280 dan komposisi gradien vs volume elusi direcord Peak elusi dari papain ditentukan menggunakan papain standar Persentase luas peak papain didapatkan dari automatic integrator Kemurnian papain diketahui dari persentase luas peak papain berbanding luas peak total

13 Analisis aktifitas enzim papain
100 µl larutan enzim dipreparasi dahulu dengan dicampurkan 200 µl 50 mM cysteine-20 mM EDTA dan 700 µl 50 mM Tris-HCl buffer pada pH 8 Campuran tersebut diinkubasi selama 5 menit pada suhu 37°C direaksikan dengan 1 ml 1% (w/v) larutan casein Setelah 10 menit, reaksi dihentikan dengan menambahkan 3 ml 5% (v/v) trichloroacetic acid (TCA) pada larutan tersebut dan didinginkan selama 1 jam Campuran itu kemudian disentrifugasi dan diambil supernatannya untuk diukur absorbansinya pada 275 nm Satuan unit aktifitas protease tersebut diukur berdasarkan jumlah enzim yang memberikan produk digesti terlarut yang menghasilkan kenaikan absorbansi pada 275 nm setara dengan 1 µmol dari tyrosine/min pada kondisi percobaan

14 Papain assay Gel Elektrophoresis katode-polyacryamide
Fig. 1. Cathodic polyacrylamide gel electrophoresis of papain during purifica- tion by two-step salt precipitation and extraction in aqueous two-phase system consisting of 8% PEG–15% (NH4)2SO4. The samples in the different lanes represent: standard papain (lane 1, 6 g), crude latex extract (lane 2), purified papain from a two-step salt precipitation (lane 3), and from top phase of the sys- tem consisting of 8% (w/w) PEG–15% (w/w) (NH4)2SO4 (lane 4), and bottom phase of the same two-phase system (lane 5) after partitioning of latex. Standar Papain Crude Latex Salt precipitate 4. Fraksi atas 2 phase aqueous 5. Fraksi bawah Bottom

15 Salt precipitation

16 Aqueous two-phase system

17 Hasil Papain Papain murni didapatkan pada fase atas FPLC dengan 1/4 volume dari keseluruhan Optimal precipitation dan two phase sistem purity (100% versus ∼89%) dari papaya latex. (top phase volume=6ml bottom phase volume = 25.5 ml).

18 FPLC 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 [µ] 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 ( psi). Thus, if the pressure of HPLC can be limited, each FPLC column may also be used in an HPLC machine.

19 Estimasi Kasar Ekonomi Analisis
Harga peralatan Analisis FPLC : $3500 Gel Elektrophoresis katode-polyacryamide : $2500 Harga papain 37.5 euro/ 225 gr Ekonomi dianalisis lebih lanjut. Coba dicari untuk lebih ada harga tiap alat dan bahannya lagi.

20 Ekonomi Analisis Papain Enzyme Dietary Supplement Concentrate The product is standardized 400 MCU/ gram Bulk Food Grade USP Powder Packaging: 225 grams Price $37.25 ea.

21 Spesifikasi Papain Sigma Alderich
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:λmax: 278 nm Extinction coefficient, E1% = 25 Extinction coefficient, EmM = 57.6 (at 280 nm) Definisi Unit: 1 unit menghidrolisis 1.0 µmole of N-α-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 : Temperature Optimum : 65 °C Sigma Alderich