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ISOLASI DAN IDENTIFIKASI AWAL SENYAWA INHIBITOR RNA HELIKASE VIRUS HEPATITIS C DARI EKSTRAK BUAH MANGROVE Avicennia marina (Forsk.) Vierh Mustopa, Apon Zaenal; ., Melki; Kusumawati, Ika Sari
Jurnal Pengolahan Hasil Perikanan Indonesia Vol 15, No 2 (2012): Jurnal Pengolahan Hasil Perikanan Indonesia
Publisher : Departement of Aquatic Product Technology

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Abstract

Hepatitis C virus is the cause of hepatitis C disease which has high virulence. Recent therapy using combination of ribavirin and alpha interferon has short efficiency (< 80%). Thus, the discovery of new drug is needed. Antiviral drugs can be discovered through molecular target therapy by finding the inhibitor of RNA helicase that play role in viral replication. Inhibitor can be derived from chemical compound produced by mangrove. The aim of this research was to isolate the active compound groups from fruit of  Avicennia marina (Forsk) which had inhibitory activity against RNA helicase. Inhibitory activity was measured by releasing of phosphate inorganic in  colorimetric ATPase assay. Crude extract was fractionated using gel filtration chromatography with methanol in chloroform solvent. The result showed that fraction 2 has the highest inhibitory activity i.e. 81.78%. Phytochemical test of crude extract indicated positive result for flavonoids, alkaloids, steroid dan triterpenoid, tannin and saponins. Moreover, high performance liquid chromatography (HPLC) analysis showed absorption peak with the high abundance at the retention time of  7.250; 18.983 and 20.050 minute at 216 nm, 247 nm and 263 nm, respectively. According to the results of phytochemical, TLC, and HPLC analyses, inhibitor compound from fruit of  A. marina (Forsk) was suggested it belongs to flavonoids.Key words: Avicennia marina, flavonoid, hepatitis C virus, RNA helicase
Identification of Antibiotic-Resistance Genes from Lactic Acid Bacteria in Indonesian Fermented Foods SUKMARINI, LINDA; MUSTOPA, APON ZAENAL; NORMAWATI, MARIDHA; MUZDALIFAH, IKRIMAH
HAYATI Journal of Biosciences Vol 21, No 3 (2014): September 2014
Publisher : Bogor Agricultural University, Indonesia

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Abstract

Lactic acid bacteria (LAB) are known to have an important role in food fermentation and are thought to have health-promoting abilities such as probiotic properties. In this study, LAB were isolated from Indonesian fermented foods such as dadih (fermented buffalo milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice). Those isolates were investigated for their resistance to two antibiotics: chloramphenicol and erythromycin. Recent efforts in food science have sought to identify genetic markers for antibiotic resistance within LAB strains, so that these genes can be selected for genetic modification. Such research is presently being directed toward the development of food-grade vectors (plasmid). The aim of this study is to screen LAB isolated from Indonesian traditional fermented foods, for chloramphenicol and erythromycin resistance. In this study, a total of 120 LAB samples were taken from traditional Indonesia fermented foods, and were tested for resistance to chloramphenicol and erythromycin. The results show that three LAB strains remained resistant to doses of up to 5 μg/mL chloramphenicol, while the LAB strain Lactobacillus plantarum showed resistance to the antibiotic erythromycin up to a concentration of 15 μg/mL.
Diversity of Lactic Acid Bacteria Isolated from Indonesian Traditional Fermented Foods MUSTOPA, APON ZAENAL; FATIMAH, FATIMAH
Microbiology Indonesia Vol 8, No 2 (2014): June 2014
Publisher : Indonesian Society for microbiology

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Abstract

The diversity of lactic acid bacteria was evaluated from Indonesian fermented foods such as dadih (buffalo fermented milk), tempoyak (fermented durian), bekasam (fermented meat), and tape ketan (fermented glutinous rice). Lactic acid bacteria were enumerated using selective media and characterised based on a genotypic methods such as rep- PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Forty-six colonies had successfullybeen isolated from Indonesian fermented foods. The great majority of these colonies originated from dadih (43.48%), tempoyak (39.13%), bekasam (13.04%) and tape (4,3%). The 46 isolates were characterised based on a genotypic methods such as RAPD and rep-PCR as well as 16S rRNA gene sequencing of representative strains. The rep-PCR result yielded seven clusters (I-VII) at a similarity level of 75-88% and RAPD-PCR used LB2 primer, M13 primer and primer A, B, C. The RAPD result using LB2 primer yielded eight clusters (I-VIII) at a similarity level of 82-91%. Identification using 16S rRNA showed that the majority strains as Lactobacillus plantarum, Lactobacillus fermentum and Pediococcus pentosaceus strains.
Characterization of chaperone-like activity of small heat shock protein (sHSP) isolated from Indonesian Traditional Food (Tempoyak ) Lactobacillus plantarum U10 HASLIA, MARGARETA; MUSTOPA, APON ZAENAL; BUDIARTO, BUGI RATNO; WIDYASTUTI, UTUT
Microbiology Indonesia Vol 8, No 4 (2014): December 2014
Publisher : Indonesian Society for microbiology

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Abstract

The characterization of small heat shock protein (sHSP) from tempoyak-originated Lactobacillus plantarum was investigated. The heat adaptive response proteins were ranging from 18 kDa to 51 kDa. Interestingly, the Intercellular Protein (IP) fraction of heat shocked-L.plantarum U10 exhibited chaperone like activity by the ability to prevent loss of proteinase K activity from denaturation. Furthermore, The sHSP gene that related to the predicted sHSP ±18 kDa protein were successfully identified by PCR method and this gene has 423 bp size. The sHSP gene has 140 amino acids (with unique motive at C-terminus T-L-P-K amino acid sequence) and has closely 100% identity with those L.plantarum isolated from food or non-food environment. Moreover, the gene encoding sHSP ±18 kDa protein was indeed up-regulated after L.plantarum U10 treated by heat shocking as proven by Reverse Transcriptase-PCR. This result suggested that sHSP ±18 kDa in our study may confers a survival advantage on Lactobacillus plantarum and capable of protecting the cell against under temperature stress.
Identification and characterization of virulence factor of several Indonesian Xanthomonas oryzae pv. oryzae FATIMAH, FATIMAH; MUSTOPA, APON ZAENAL; KUSNANDARSYAH, IQBAL
Microbiology Indonesia Vol 8, No 3 (2014): September 2014
Publisher : Indonesian Society for microbiology

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Abstract

Xanthomonas oryzae pv. oryzae (Xoo) is the bacterial causative agent of leaf blight in rice (Oryza sativa L.), the most serious bacterial disease of rice in many rice growing areas worldwide. This study aimed to identify and characterize several virulence factors of seven Xoo isolates from Yogyakarta, West Java, and West Sumatera. The identification of Xoo using 16S rRNA confirmed high homology to Xanthomonas oryzae pv. oryzae PXO99A and revealed three groups. The first group was Xoo93229, the second group containing Xoo1110, Xoo1122, Xoo1130, Xoo7624 and Xoo8024 as the same cluster with PXO99A and the third group was KACC10331 and MAFF311018. The amounts of exopolysaccharide (EPS) and cellulase produced were varying depending on the Xoo isolates. The EPS were produced more by isolate Xoo1130, Xoo1122 and Xoo8024. All tested isolates revealed similar cellulase activity except for isolate Xoo8024. The pathogenicity assay among the Xoo isolate showed that all tested isolates were virulent except Xoo7624. The in planta assay revealed that the tested isolates have multiplied and continued increasing the population size except for Xoo1110 and Xoo7624. High yield of EPS, cellulase activity, more virulence, and increasing population size revealed from isolate Xoo1130 and Xoo1122.
CONSTRUCTION, EXPRESSION AND PURIFICATION OF RECOMBINANT PRE-MATURE PEPTIDE OF PLANTARICIN F FROM Lactobacillus plantarum S34 IN Escherichia coli Kusdianawati, Kusdianawati; Mustopa, Apon Zaenal; Suharsono, Suharsono; Budiarto, Bugi Ratno; Fatimah, Fatimah; Danuri, Hasim
Indonesian Journal of Agricultural Science Vol 16, No 1 (2015): April 2015
Publisher : Indonesian Center for Agricultural Library Technology Dissemination - IAARD

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Abstract

Plantaricin is one of bacteriocins that have the potential to be used as food preservative. Plantaricin is safe for human consumption because it can be easily degraded by proteolytic enzymes. The objective of this study was to express and purify recombinant pre-mature peptide of plantaricin F from Lactobacillus plantarum S34 in Escherichia coli. Plantaricin gene-specific primer was used to obtain pln F structural gene amplicon from L. plantarum S34. This amplicon was cloned in pET32a vector and expressed in E. coli BL21 (DE3) pLysS. Pre-mature plantaricin F peptide was expressed as Histagged-fusion protein and separated by Co2+-chelating affinity chromatography. L. plantarum S34-derived pre-mature plantaricin F peptide fused with thioredoxin-(His)6tag had successfully been expressed in E. coli BL21 (DE3) pLysS using pET32a as an expression vector. The fused recombinant pln F as pre-mature state expressed had a molecular mass of +24 kDa, meanwhile the fused recombinant that contained only the leader peptide of pln F appeared as +20 kDa based on SDS-PAGE separations. The optimal production of fused recombinant pln F as soluble fraction was obtained when culture condition was added with 0.5 mM of IPTG and incubated at 22°C for 5 hours (OD~1). Furthermore, the expression of fused recombinant pln F as its pre-mature peptide pointed out that the pln F’s leader peptide could be proteolytically cleaved by a system in heterologous cells. Overall, heterologous pln F production as pre-mature peptide fused with thioredoxin-(His)6tag had been well established. From this research, we expect plantaricin F can be expressed and purified in E. coli.
ANTIBACTERIAL ACTIVITY ASSAY OF MANGROVE EXTRACTS AGAINST SALMONELLA TYPHI AND LISTERIA MONOCYTOGENES Mustopa, Apon Zaenal; Umami, Rifqiyah Nur; Melki, .
Jurnal Ilmu dan Teknologi Kelautan Tropis Vol 7, No 2 (2015): Elektronik Jurnal Ilmu dan Teknologi Kelautan Tropis
Publisher : Jurnal Ilmu dan Teknologi Kelautan Tropis

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Abstract

The antibacterial activities of mangrove species, Avicennia marina, Sonneratia caseolaris (collected from Teluk Payo, Banyuasin, South Sumatera), Ceriops tagal, Rizhopora apiculata, and Sonneratia alba (collected from Sadai, South Bangka) were screened against Salmonella typhi and Listeria monocytogenes by agar disk diffusion assays. Extractions were conducted using organic solvents (methanol, ethyl acetate, and acetone, subsequently). Most of the extracts tested showed potential antibacterial activity against both pathogens. The methanol extracts of the bark from S. alba and the fruit from A. marina showed particularly large inhibition zones (15 mm) against S. typhi. The acetone extract of S. alba leaves showed the largest inhibition zone (14 mm) when tested against L. Monocy-togenes. Further partial purifications of selected extracts which showed strong inhibition were performed by silica gel column chromatography using various eluent compositions with different polarities. The third fraction of methanol extract from S.alba leaves eluted with chloroform:methanol (1:5) resulted in a remarkably large inhibition zone (23 mm) against S. typhi. The third and seventh fractions of acetone extract from S. alba leaves eluted with ethyl acetate:methanol (7:3) resulted in a large inhibition zones (15 mm) against L. monocytogenes. In addition, the sixth fraction of methanol extract from A. marina fruit eluted with chloroform : methanol (6:4) resulted in the largest inhibition zone (17 mm) against L. monocytogenes. These results indicated that mangrove extracts could be developed as potential biomaterials for biopharmaceutical as well as biopreservation industries.Keywords: antibacterial activity, mangrove, column chromatography
Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity Solikhin, Akhmad; Mustopa, Apon Zaenal; Suharsono, Suharsono; Putranto, Wendry Setiyadi
ANNALES BOGORIENSES Vol 22, No 2 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

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Abstract

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.
Molecular Identification of Microalgae BTM 11 and its Lectin Isolation, Characterization, and Inhibition Activity Mustopa, Apon Zaenal; Isworo, Rhestu; Nurilmala, Mala; Susilaningsih, Dwi
ANNALES BOGORIENSES Vol 20, No 2 (2016): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

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Abstract

BTM 11 is unknown species of microalgae, but has active compounds that can inhibit viruses. One of proteins produced by microalgae is a lectin. Lectin is a carbohydrate-binding protein found in various microalgae that show antiviral and antibacterial activity. The purpose of this study was to perform identification of the species of microalgae BTM 11, isolation, characterization, and assay of lectin inhibitory activity. Microalgae BTM 11 was identified to have homology with Cyanobacterium 99% and Geitlerinema sp 98%. Lectin of microalgae BTM 11 was isolated by ammonium sulfate precipitation of 75% with a molecular weight of 17 kDa. Lectin protein activity of microalgae BTM 11 was able to inhibit the enzyme activity of RNA helicase hepatitis C by 57.90% and 27.55%. In addition, the protein was able to suppress the activity of Staphylococcus aureus ATCC 6538, E. coli EPEC K.1.1. and Salmonella typhii ATCC 25241. Activitiy of lectin was stable at 30 °C and was unaffected by the action of the enzyme. These results indicate that lectin of microalgae BTM 11 could be a alternative to antiviral and antibacterial proteins.
APLIKASI MARKA MOLEKULER PADA BUAH DAN BIJI KOPI ASAL KALIMANTAN TIMUR [Molecular marker application of cherry and green bean of East Kalimantan coffee] Fatimah, Fatimah; Urnemi, Urnemi; Mustopa, Apon Zaenal; Syahrumsyah, Hudaida
BERITA BIOLOGI Vol 13, No 1 (2014)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

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Abstract

Two quantitative traits, cherry and green bean characters are the important phenotypic selection in coffee breeding practice. The important well-known character from coffee markets is cherry and bean size. In this study, 43 genotypes of coffee were collected from four districts in East Kalimantan i.e. Kutai Kertanegara, Kutai Timur, Berau dan Paser Utara. The objective of this study was to identify cherry and green bean character using quantitative trait locus (QTL) molecular marker, genetic variation from developed alleles, cluster analysis and association analysis of molecular marker, and phenotype observation. Based on polymorphic information content (PIC) of primers used in this study, the genetic variation was low. Based on cluster analysis, two major groups were identified. The first group corresponds to Arabika that consisted of 3 districts, Kutai Timur, Berau and Paser Utara. The second group correspond to Robusta mostly from Kutai Kertanegara.Significant association of primer markers M480 and M312 with QTL has suggested that they can be used as specific primers linked to size of cherry and green bean.Furthermore,they were potential marker assisted breeding in coffee breeding program.