PEMURNIAN POLISAKARIDA DARI MIKROALGA BTM 11 SEBAGAI INHIBITOR RNA HELIKASE VIRUS HEPATITIS C

Infeksi virus hepatitis C (HCV) merupakan penyebab utama penyakit hati kronis di seluruh dunia. Polisakarida dari mikroalga memiliki kemampuan melawan infeksi HCV. Ekstrak mikroalga BTM 11 mampu melawan HCV dengan cara menghambat RNA helikase. Penelitian ini ditujukan untukmemurnikan polisakarida dari ekstrak mikroalga BTM 11. Tahapan penelitian ini meliputi isolasi RNA helikase HCV dari bakteri Escherichia coli BL21 (DE3) pLyss yang membawa gen NS3 helikase dalam plasmid pET 21b menggunakan kromatografi afinitas, dan pemurnian polisakarida dari mikroalga BTM 11 dengan kromatografi gel filtrasi dan ion-exchange. Aktivitas inhibitor diketahui dari besarnya aktivitas ATPase yang dimiliki oleh RNA helikase. Analisis SDS-PAGE menunjukkan RNA helikase terpurifikasi dengan bobot molekul 54 kDa. Kromatografi gel filtrasi menghasilkan fraksi aktif polisakarida inhibitor RNA helikase dengan aktivitas sebesar 78,76% dan kandungan gula sebesar 2,97 mg/mL, sedangkan kromatografi ion-exchange memiliki aktivitas polisakarida inhibitor sebesar 74,6% dengan kandungan gula sebesar 3,21 mg/ mL. Profil inhibitor pada kromatografi lapis tipis (KLT) menunjukkan satu spotsenyawa aktif, namun hasil kromatografi cair kinerja tinggi (KCKT) diperoleh tiga puncak senyawa yang menunjukkan bahwa hasil fraksi polisakarida inhibitor belum murni

PURIFICATION AND CHARACTERIZATION OF POLYSACCHARIDE FROM MICROALGAE BTM 11 AS INHIBITOR OF HEPATITIS C VIRUS RNA HELICASE

Hepatitis C virus is one of the causative agents for HCV-related liver disease development with high virulence. Antiviral drugs can be discovered through molecular target-based therapy by finding the inhibitors for RNA helicase that play crucial role in viral replication. An inhibitor can be derived from polysaccharides produced by microalgae. In this study, polysaccharide from microalgae BTM11 which had inhibitory activity against RNA helicase have been purified and characterized.On the other hand, the RNA helicase was produced by E. coli BL21(DE3)pLyss harboring NS3 RNA helicase HCV gene in pET-21b plasmid. This enzyme then was purified by affinity chromatography and this purified enzyme was used for HCV RNA helicase inhibitory assay. Polysaccharide fractions were separated from the extract of BTM 11 using Sepharose 4B column chromatography. Inhibitor activity was measured using colorimetry ATPase assay based on releasing of phosphate inorganic. The results of SDS-PAGE and Western blot showed that the purified RNA helicase had a molecular weight of 54kDa. The highest inhibition activity of HCV RNA helicase (88 ± 2,4726%) was achieved at fraction 10 of purified polysaccharide. The HPLC result showed that compounds of polysaccharide active fraction were maltopentose (Rt 4.183) and glucose (Rt 5.673). Both of 1H-NMR and IR spectra showed hydroxyl and carbonyl groups that present in the polysaccharide structure

PURIFICATION AND CHARACTERIZATION OF POLYSACCHARIDE FROM MICROALGAE BTM 11 AS INHIBITOR OF HEPATITIS C VIRUS RNA HELICASE

Hepatitis C virus is one of the causative agents for HCV-related liver disease development with high virulence. Antiviral drugs can be discovered through molecular target-based therapy by finding the inhibitors for RNA helicase that play crucial role in viral replication. An inhibitor can be derived from polysaccharides produced by microalgae. In this study, polysaccharide from microalgae BTM11 which had inhibitory activity against RNA helicase have been purified and characterized.On the other hand, the RNA helicase was produced by E. coli BL21(DE3)pLyss harboring NS3 RNA helicase HCV gene in pET-21b plasmid. This enzyme then was purified by affinity chromatography and this purified enzyme was used for HCV RNA helicase inhibitory assay. Polysaccharide fractions were separated from the extract of BTM 11 using Sepharose 4B column chromatography. Inhibitor activity was measured using colorimetry ATPase assay based on releasing of phosphate inorganic. The results of SDS-PAGE and Western blot showed that the purified RNA helicase had a molecular weight of 54kDa. The highest inhibition activity of HCV RNA helicase (88 ± 2,4726%) was achieved at fraction 10 of purified polysaccharide. The HPLC result showed that compounds of polysaccharide active fraction were maltopentose (Rt 4.183) and glucose (Rt 5.673). Both of 1H-NMR and IR spectra showed hydroxyl and carbonyl groups that present in the polysaccharide structure.      Key words: Hepatitis C Virus, RNA Helicase, Microalgae BTM11, chromatography, polysaccharid