Khairunnisa’, Amalia and Prof. Dr. Ir. Abdul Latief Abadi,, MS and Muhammad Akhid Syib’li,, SP., MP., Ph.D (2022) Studi Kandidat Senyawa Alami Antifungi Dalam Menghambat Patogen Fusarium Sp. Secara In Silico Dan In Vitro. Sarjana thesis, Universitas Brawijaya.
Abstract
Jamur Fusarium graminearum (Fg) penyebab Fusarium Head Blight (FHB) pada gandum dan Fusarium oxysporum f.sp. cepae (Foc) penyebab penyakit moler pada bawang merah mengakibatkan penurunan produksi hingga >50%. Petani masih bergantung pada antifungi fungisida kimia sintetik dalam pengendaliannya, namun cenderung kurang efektif dan menyebabkan resistensi patogen. Antifungi yang lebih efektif sangat diperlukan namun biaya dalam proses perancangan antifungi baru $256 juta selama >10 tahun, mahal dan lama (Lamberth et al., 2013). Bioinformatika melalui metode Computer-Aided Drug Design dapat menjadi solusi untuk merancang senyawa yang berpotensi sebagai bahan aktif antifungi secara in silico (Salmaso & Moro, 2018). CADD memprediksi interaksi antara protein target dengan senyawa dan mengidentifikasi struktur senyawa yang sesuai untuk mengikat protein target myosin-I (Fg) dan kutinase (Foc). Penelitian mengenai penghambatan senyawa terhadap patogen Fusarium sp. dilakukan secara in vitro. Penelitian ini bertujuan untuk mengetahui nilai molecular docking hasil in silico kandidat bahan aktif antifungi serta mengobservasi pengaruh senyawa alami terpilih dalam menghambat patogen Foc dan aktivitas kutinasenya. Penelitian ini dilakukan dari Oktober 2021-Mei 2022 di Lab. Pengendalian Hayati, Dep. Hama dan Penyakit Tumbuhan, Fakultas Pertanian, Universitas Brawijaya. Tahapan penelitian dimulai dari uji in silico (analisis pustaka biologi myosin-I dan kutinase, preparasi protein dan senyawa, validasi struktur protein, dan molecular docking). Uji in vitro meliputi sterilisasi alat, pembuatan media jamur, purifikasi dan identifikasi jamur Foc, pembuatan slide culture, media uji kutinase, dan ekstrak senyawa alami, serta uji aktivitas dan penghambatan enzim kutinase. Parameter pengamatan pada penelitian ini ada dua, yaitu in silico dan in vitro. Pada uji in silico terdapat binding energy dan konstanta inhibisi (Ki). Uji in vitro meliputi penghambatan pertumbuhan jamur (diameter) dan penghambatan aktivitas enzim kutinase (efek fluoresensi oranye). Analisis in silico dilakukan melalui visualisasi hasil interaksi antara protein dan senyawa secara 3D dan 2D. Pada in vitro, diameter pertumbuhan jamur dianalisis secara statistik menggunakan anova sedangkan efek fluoresensi oranye dilakukan secara kualitatif. Hasil penelitian menunjukkan bahwa kandidat senyawa terhadap protein target memiliki nilai molecular docking rata-rata -6.6185 dan -3.951 kcal/mol. Pada uji penghambatan pertumbuhan jamur Foc dan aktivitas enzim kutinase, perlakuan kontrol berbeda nyata dengan perlakuan lainnya dan menghasilkan efek fluoresensi oranye terbanyak. Senyawa terpilih mampu menghambat aktivitas kutinase Foc. Kurkumin dapat direkomendasikan sebagai bahan aktif antifungi. Penelitian aktivitas enzim kutinase pada jamur Foc secara kuantitatif menggunakan spektrofotometer diperlukan guna memperoleh data yang lebih akurat dan spesifik. Penelitian secara in vivo juga diperlukan untuk mengetahui efek penghambatan senyawa secara langsung
English Abstract
Fusarium graminearum (Fg) that causes Fusarium Head Blight (FHB) on wheat and Fusarium oxysporum f.sp. cepae (Foc) causes moler disease in shallots resulting in a decrease in production >50%. Farmers still depend on synthetic chemical antifungal fungicides in their control, but these tend to be less effective and cause pathogen resistance. More effective antifungals are urgently needed but the cost of designing an antifungal is only $256 million over >10 years (Lamberth et al., 2013). Bioinformatics through the Computer-Aided Drug Design method can be a solution for designing compounds that have the potential as antifungal active ingredients in silico (Salmaso & Moro, 2018). CADD predicts the interaction between the target protein and the compound and identifies the structure of the appropriate compound to bind to the target proteins myosin-I (Fg) and cutinase (Foc). Research on the inhibition of compounds against the pathogen Fusarium sp. carried out in vitro. This study aims to determine the value of in silico molecular docking of candidate antifungal active ingredients and to observe the effect of selected natural compounds in inhibiting Foc pathogens and their cutinase activity. The research was conducted from October 2021-May 2022 in the Lab. Biological Control, Dept. Pests and Plant Diseases, Faculty of Agriculture, Universitas Brawijaya. The research stages were started from in silico test (analysis of myosin-I and cutinase biology literature, preparation of proteins and compounds, validation of protein structure, and molecular docking). In vitro tests include sterilization of equipment, manufacture of fungal media, purification and identification of Foc, manufacture of slide cultures, cutinase test media, and extracts of natural compounds, as well as test the activity and inhibition of the enzyme cutinase. There are two observation parameters in this study, namely in silico and in vitro. In the in silico test, there are binding energy and inhibition constants (Ki). In vitro tests included inhibition of fungal growth (diameter) and inhibition of cutinase enzyme activity (orange fluorescence zone). In silico analysis was carried out by visualizing the interaction results between proteins and compounds in 3D and 2D. In vitro, the diameter of fungal growth was statistically analyzed using ANOVA while the orange fluorescence zone was analyzed qualitatively. The results showed that the candidate compound against the target protein had an average molecular docking value of -6.6185 and -3.951 kcal/mol. In the Foc fungal growth inhibition test and cutinase activity, the control treatment was significantly different from the others and produced the most orange fluorescence zones. Selected compound was able to inhibit the activity of cutinase Foc. Curcumin can be recommended as an antifungal active ingredient. Quantitative research on the activity of the cutinase enzyme in Foc mushrooms using a spectrophotometer to obtain more accurate and specific data. In vivo research is also needed to determine the direct inhibitory effect of the compound.
Other obstract
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| Item Type: | Thesis (Sarjana) |
|---|---|
| Identification Number: | 0522040163 |
| Subjects: | 300 Social sciences > 338 Production > 338.1 Agriculture > 338.16 Production efficiency |
| Divisions: | Fakultas Pertanian > Agroekoteknologi |
| Depositing User: | Nur Cholis |
| Date Deposited: | 15 Nov 2022 02:49 |
| Last Modified: | 15 Nov 2022 02:49 |
| URI: | http://repository.ub.ac.id/id/eprint/196325 |
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