Skrining Actinomycetes yang Berpotensi Sebagai Inhibitor Enzim 3-Chymotripsin-like Protease (3CLpro) SARS-CoV 2

Mihardja, Joshua Nicholas and Tunjung Mahatmanto, STP., M.Si., Ph.D and DR. Is Helianti, M.Sc (2024) Skrining Actinomycetes yang Berpotensi Sebagai Inhibitor Enzim 3-Chymotripsin-like Protease (3CLpro) SARS-CoV 2. Sarjana thesis, Universitas Brawijaya.

Abstract

SARS-CoV 2 merupakan suatu virus yang menyebabkan penyakit COVID-19, suatu jenis penyakit pernapasan yang pertama kali dilaporkan pada 2019 dan menjadi pandemi globa sejak saat tersebut. Berbagai penelitian terkait pembentukan obat COVID-19 telah menghasilkan vaksin dan juga antiviral yang secara spesifik didesain untuk menargetkan enzim 3CLpro, yakni enzim yang memediasi siklus infeksi virus tersebut. Penelitian terkait antiviral SARS-CoV 2 saat ini didominasi penemuan senyawa sintetis kimiawi, akan tetapi dewasa ini trend penelitian tersebut mulai berfokus pada penemuan senyawa alami yang didapat dari makhluk hidup. Penelitian-penelitian in silico terdahulu menemukan metabolit sekunder Actinomycetes memiliki aktivitas inhibisi terhadap aktivitas enzim 3CLpro, akan tetapi belum ada penelitian lebih lanjut untuk membuktikan hal tersebut. Penelitian ini dilakukan untuk menemukan kandidat potensial inhibitor enzim 3CLpro dari ekstrak mikroba spesies Actinomycetes melalui High Throughput Screening yang mengimplementasikan fluorescence-based enzyme assay. Penelitian ini meliputi produksi enzim 3CLpro rekombinan melalui sistem ekspresi E.coli BL21 DE3, assay 1st screening kandidat potensial, assay rekonfirmasi terhadap ekstrak kandidat, dan penentuan IC50 ekstrak. Proses produksi enzim 3CLpro menghasilkan enzim terpurifikasi dengan variasi konsentrasi dari 1.945 mg/ml (batch 1) hingga 3.021 mg/ml (batch 2). Enzim tersebut kemudian digunakan sebagai target inhibisi dalam uji selanjutnya. 1st screening telah menemukan 29 kandidat potensial namun hanya 5 spesies yang memiliki aktivitas inhibisi yang konsisten dengan 1st screening setelah diuji rekonfirmasi. Dari 5 spesies tersebut, dipilih dua spesies untuk diuji lanjut, yakni Streptomyces tritici dan Nocardia aobensis. Proses uji selanjutnya berhasil menemukan ekstrak Streptomyces tritici memiliki aktivitas inhibisi yang lebih tinggi, sebesar 94 hingga 114%, dibandingkan dengan ekstrak Nocardia aobensis, sebesar 39 hingga 65%. IC50 dari ekstrak Streptomyces tritici adalah sebesar 43.16 ppm sedangkan ekstrak Nocardia aobensis sebesar 94.38 ppm

English Abstract

SARS-CoV 2 is a virus that causes COVID-19, a form of respiratory system disease that was first reported in 2019 and managed to become a global pandemic ever since. Multiple researches towards finding a cure for COVID-19 has resulted the creation of vaccines and antivirals that specifically target 3CLpro enzyme, an enzyme crucial to the virus’ life cycle, to combat SARS-CoV2. SARS-CoV2 antiviral research is currently dominated with synthetic chemically derived compounds, in which current research trends has started to shift towards bio-compounds derived from living organisms. Past in silico researches have shown compounds derived from Actinomycetes species to possess inhibitory activities towards 3CLpro, but such researches have yet to be further proven. This study aims to discover potential inhibitor candidates of 3CLpro enzyme from Actinomycetes species extract through High Throughput Screening that implements fluorescence-based enzyme assay. This research includes the production of recombinant 3CLpro enzyme through BL21 DE3 E. coli expression system, 1st screening of potential candidates, reconfirmation tests of potential candidates, and the determination of extract IC50. Production of 3CLpro resulted in high purity enzyme with concentration varying from 1.945 mg/ml to 3.021 mg/ml. Such enzyme is then used as inhibition targets in the following steps. 1st screening test has shown 29 potential candidates in which only 6 species extract has shown similar results compared to 1st screening. Further test is conducted to 2 out of 6 species, which are Streptomyces tritici and Nocardia aobensis. Further tests shown that Streptomyces tritici possessing the higher inhibitory activities, at 94 to 114% inhibition, compared to Nocardia aobensis, at 39 to 65% inhibition. The IC50 of Streptomyces tritici is determined at 43.16 ppm while Nocardia aobensis is determined at 94.38 pp

Item Type:	Thesis (Sarjana)
Identification Number:	052410
Divisions:	Fakultas Teknologi Pertanian > Keteknikan Pertanian
Depositing User:	soegeng sugeng
Date Deposited:	14 Aug 2024 03:02
Last Modified:	14 Aug 2024 03:02
URI:	http://repository.ub.ac.id/id/eprint/223869

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