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Isolasi, Purifikasi, Identifikasi, Dan Optimasi Medium Fermentasi Antibiotik Yang Dihasilkan Oleh Aktinomisetes Laut

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Isolasi, Purifikasi, Identifikasi, Dan Optimasi Medium Fermentasi Antibiotik Yang Dihasilkan Oleh Aktinomisetes Laut ISOLASI, PURIFIKASI, IDENTIFIKASI, DAN OPTIMASI MEDIUM FERMENTASI ANTIBIOTIK YANG DIHASILKAN OLEH AKTINOMISETES LAUT ROFIQ SUNARYANTO SEKOLAH PASCASARJANA INSTITUT PERTANIAN BOGOR BOGOR 2011 PERNYATAAN MENGENAI DISERTASI DAN SUMBER INFORMASI Dengan ini saya menyatakan bahwa disertasi dengan judul ”Isolasi, Purifikasi, Identifikasi, dan Optimasi Medium Fermentasi Antibiotik yang Dihasilkan oleh Aktinomisetes Laut” adalah karya saya dengan arahan dari komisi pembimbing dan belum diajukan dalam bentuk apapun kepada perguruan tinggi manapun. Sumber informasi yang berasal atau dikutip dari karya yang diterbitkan maupun tidak diterbitkan dari penulis lain telah disebutkan dalam teks dan dicantumkan dalam Daftar Pustaka di bagian akhir disertasi ini. Bogor, Juli 2011 Rofiq Sunaryanto NIM. F361070142 ABSTRACT ROFIQ SUNARYANTO. Isolation, Purification, Identification, and Fermentation Medium Optimization of Antibiotic Produced by Marine Actinomycetes. Under direction of TUN TEDJA IRAWADI, ZAINAL ALIM MAS’UD, LIESBETINI HARTOTO, BAMBANG MARWOTO. Isolation and purification of active compounds produced by marine actinomycetes has been carried out. Marine sediment samples were obtained from 3 different places in Banten West Coast, Cirebon North Coast, and Yogyakarta South Coasts. A total of 40 actinomycetes isolates were obtained 4 isolates were active against Escherichia coli ATCC 25922, 5 isolates were active against Staphylococcus aureus ATCC25923, 4 isolates were active against Bacillus subtilis ATCC 66923, 4 isolates were active against Pseudomonas aeroginosa ATCC27853, 4 isolates were active against Candida albican BIOMCC00122, and 4 isolates were active against Aspergillus niger BIOMCC00134. A11 isolate showed the most active to Gram-positive and Gram-negative bacteria. Species identification using 16S rRNA gene sequencing showed that A11 isolate is Streptomyces sp. Elucidation of its molecular formula and structure using LC-MS, 1H NMR, 13C NMR, and 13C DEPT NMR showed the antibiotic was cyclo(tyrosyl-prolyl), molecule formula was C14H16N2O3 which has a melting point of 140 °C. Minimum Inhibitory Concentration (MIC) of the antibiotic was determined against 4 bacterial test strains, namely Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 66923, which were inhibited at 27, 69, 80, and 74 µg mL-1, respectively. Fermentation profile of Streptomyces sp. A11 showed a lag phase which occurred until 8 hours, a log phase from 9 until 48 hours and a stationary phase from 48 until 144 hours. The growth phase showed maximum specific growth rate -1 (μmax) of 0.04 hour and the rate of substrate conversion into biomass (Yx/s) of 0.6 gram biomass per gram substrate. The optimum temperature and pH of cyclo(tyrosyl-prolyl) fermentation were 30 °C and 6.5-7.5, respectively. Optimum composition of fermentation medium was determined with three independent variables: dextrin as a carbon source, peptone as nitrogen source, and a mixture of mineral salts using Response Surface Methodology. The results showed that the three variables significantly affected the activity of cyclo(tyrosyl- prolyl). Peptone gave the strongest effect compared to dextrin and mineral salts. Interaction was found between dextrin and peptone. On the contrary, no interaction was observed between peptone and mineral salts, and between dextrin and mineral salts. Using a mathematical model, the most optimum composition of the medium were found to be dextrin (32.55 g L-1), peptone (11.22 g L-1), and mineral salt (8.65 mL), in which 51.54 g L-1 cyclo(tyrosyl-prolyl) was produced. Verification of the model in laboratory showed the cyclo(tyrosyl-prolyl) activity to be 50.04 mg L-1. Thus, the difference between the result of the experiment and the expected response value was 2.9%. Keywords: marine actinomycetes, antibiotic, Streptomyces, cyclo(tyrosyl-prolyl). RINGKASAN ROFIQ SUNARYANTO. Isolasi, Pemurnian, Identifikasi, dan Optimasi Medium Fermentasi Antibiotik yang Dihasilkan oleh Aktinomisetes Laut. Dibawah bimbingan TUN TEDJA IRAWADI, ZAINAL ALIM MAS’UD, LIESBETINI HARTOTO, BAMBANG MARWOTO Kebutuhan antibiotik, anti fungal, maupun anti kanker baru masih sangat diperlukan, terutama yang efektif melawan bakteri resisten, virus, protozoa, fungi atau kanker. Untuk mendapatkan antibiotik baru, para peneliti telah banyak melakukan berbagai cara seperti eksplorasi senyawa aktif dari mikroba, tumbuhan, maupun sintesis secara kimia. Salah satu mikroba yang banyak diteliti untuk diambil senyawa aktifnya adalah aktinomisetes. Indonesia merupakan negara kepulauan yang memiliki bentangan laut yang luas, kurang lebih 3,1 juta km2 atau hampir 2 kali lipat dibandingkan luas daratannya. Karakteristik laut yang bermacam-macam mengindikasikan biodiversitas hayati yang besar, khususnya biodiversitas mikroba laut. Namun demikian potensi ini belum banyak dimanfaatkan. Penelitian ini bertujuan untuk mendapatkan senyawa aktif yang berpotensi sebagai antibiotik dan memproduksinya dalam skala laboratorium yang dihasilkan oleh aktinomisetes laut melalui isolasi, penapisan, pemurnian, identifikasi dan optimasi medium fermentasi. Telah dilakukan isolasi dan penapisan aktinomisetes laut yang mampu menghasilkan senyawa antibakteri. Sampel sedimen laut diambil dari 3 tempat berbeda yaitu di Pantai Barat Banten, Pantai Utara Cirebon, dan Pantai Selatan Yogyakarta. Hasil percobaan menunjukkan bahwa pra-perlakuan dengan pemanasan dan pengasaman sampel serta penambahan sikloheksimid 100 μg mL- 1, nistatin 25 μg mL-1, asam nalidiksat 20 μg mL-1, dan rifampisin 5 μg mL-1 mampu menekan pertumbuhan bakteri dan kapang kontaminan. Total hasil isolasi diperoleh dari sampel sedimen laut sebanyak 40 isolat aktinomisetes. Penapisan dengan menggunakan 6 macam mikroba uji diperoleh 4 isolat yang mampu menghambat pertumbuhan Escherichia coli ATCC 25922, 5 isolat menghambat Staphylococcus aureus ATCC25923, 4 isolat menghambat Bacillus subtilis ATCC 66923, 4 isolat menghambat Pseudomonas aeroginosa ATCC27853, 4 isolat menghambat Candida albican BIOMCC00122, dan 4 isolat menghambat Aspergillus niger BIOMCC00134. Isolat A11 menunjukkan isolat yang memiliki daya hambat paling kuat terhadap bakteri Gram-positif dan Gram-negatif sehingga isolat tersebut dipilih untuk penelitian selanjutnya. Hasil identifikasi menggunakan 16S rRNA menunjukkan bahwa isolat A11 adalah Streptomyces sp. (homology 100%) kelas Actinobacteria, ordo Actinomycetales, famili Streptomycetaceae, dan genus Streptomyces. Fermentasi isolat A11 dilakukan selama 144 jam dengan menggunakan medium khamir-pepton. Dari kaldu fermentasi diperoleh bobot kering sel, ekstrak sel dengan metanol, dan ekstrak supernatan dengan etil asetat berturut-turut sebanyak 4,73 g L-1, 2,72 g L-1, 0,33 g L-1. Hasil uji aktivitas antimikroba (bioassay) menunjukkan bahwa ekstrak aktif hanya terjadi pada ekstrak supernatannya saja, hal ini menunjukkan bahwa senyawa aktif dihasilkan secara ekstraselular. Ekstrak supernatan yang terbukti memiliki aktivitas antimikroba selanjutnya dipurifikasi dengan menggunakan kromatografi kolom dan HPLC preparatif, selanjutnya dilakukan elusidasi struktur molekulnya. Hasil analisis menggunakan LC-MS diketahui senyawa aktif yang dihasilkan oleh isolat A11 -1 memiliki bobot molekul sebesar 260 g mol , rumus molekul C14H16N2O3. Hasil elusidasi struktur molekul menggunakan 1HNMR, 13C NMR, DEPT 13C NMR, dan FTIR diketahui senyawa aktif yang dihasilkan oleh Streptomyces sp.A11 adalah siklo(tirosil-prolil). Senyawa aktif ini memiliki titik leleh sebesar 140 oC. Minimum Inhibitory Concentration (MIC) ditentukan menggunakan 4 mikroba uji dengan metode difusi agar kertas cakram. Hasil percobaan menunjukkan bahwa senyawa aktif yang dihasilkan oleh Streptomyces sp.A11 memiliki MIC terhadap Escherichia coli ATCC 25922 sebesar 27 μg mL-1, Pseudomonas aeruginosa ATCC 27853 sebesar 69 μg mL-1, Staphylococcus aureus ATCC 25923 sebesar 80 μg mL-1, and Bacillus subtilis ATCC 66923 sebesar 74 μg mL-1. Profil fermentasi isolat Streptomyces sp. menunjukkan fase lag terjadi sampai dengan jam ke-8, fase pertumbuhan cepat (fase logaritma) terjadi pada selang waktu jam ke-9 sampai dengan jam ke-48, dan fase stasioner terjadi pada selang waktu jam ke-48 sampai dengan jam ke-144. Pada fase pertumbuhan cepat -1 (fase logaritma) laju pertumbuhan maksimum (μmaks) sebesar 0,04 jam dan rendemen pembentukan biomassa per massa substrat (Yx/s) sebesar 0,6 g biomassa per massa substrat. Senyawa aktif diproduksi setelah memasuki fase stasioner yaitu mulai jam ke-60 yang menunjukkan bahwa senyawa aktif yang dihasilkan tergolong dalam metabolit sekunder. Penentuan suhu optimum pada proses fermentasi untuk menghasilkan siklo(tirosil-prolil) dilakukan dalam rentang suhu 26 sampai dengan 34 °C. Hasil percobaan menunjukkan bahwa suhu 30 °C merupakan suhu terbaik untuk proses fermentasi siklo(tirosil-prolil). Penentuan pH optimum proses fermentasi siklo(tirosil-prolil) dilakukan pada rentang pH 4 sampai dengan pH 8. Hasil percobaan menunjukkan bahwa kisaran pH 6,5 sampai dengan pH 7,5 adalah kisaran pH terbaik untuk proses fermentasi siklo(tirosil-prolil). Optimasi medium fermentasi dilakukan dengan menggunakan Response Surface Methodology. Variabel bebas yang digunakan adalah dekstrin sebagai sumber karbon, pepton sebagai sumber nitrogen, dan mineral. Respon yang ditentukan adalah konsentrasi siklo(tirosil-prolil). Hasil penelitian menunjukkan bahwa ketiga variabel bebas yang digunakan dalam proses optimasi medium fermentasi ini
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