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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

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.21082/ijas.v16n1.2015.31-38

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 OF EXTRACELLULAR PROTEASE ISOLATED FROM AN ALGICOLOUS FUNGUS XYLARIA PSIDII KT30 AGAINST GRAM-POSITIVE BACTERIA Indarmawan, Taufik; Mustopa, Apon Zaenal; Budiarto, Bugi Ratno; Tarman, Kustiariyah
HAYATI Journal of Biosciences Vol. 23 No. 2 (2016): April 2016
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1051.929 KB) | DOI: 10.4308/hjb.23.2.73

Abstract

Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilisand Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate?polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.
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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (713.673 KB) | DOI: 10.4308/hjb.21.3.144

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 ?g/mL chloramphenicol, while the LAB strain Lactobacillus plantarum showed resistance to the antibiotic erythromycin up to a concentration of 15 ?g/mL.
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)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ann.bogor.2018.v22.n2.47-56

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)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v20i2.251

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

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (133.562 KB) | DOI: 10.14203/beritabiologi.v13i1.649

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.
CLONING, EXPRESSION, AND PARTIAL PURIFICATION OF PLANTARICIN W LOCUS PRODUCED BY Lactobacillus plantarum S34 [Kloning, Ekspresi, dan Purifikasi Parsial Lokus Plantarisin W Diproduksi oleh Lactobacillus plantarum S34] Umami, Rifqiyah Nur; Mustopa, Apon Zaenal; Sukmarini, Linda; Danuri, Hasim; Putri, Andini Setyanti; Wibowo, Krisna Dwi Aria
BERITA BIOLOGI Vol 16, No 1 (2017)
Publisher : Research Center for Biology-Indonesian Institute of Sciences

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (2760.052 KB) | DOI: 10.14203/beritabiologi.v16i1.2174

Abstract

Lactobacillus plantarum S34 dilaporkan mempunyai aktivitas antibakteri yang terkait dengan produksi bakteriosin. Bagian dari gen yang menyandikan salah satu lokus bakteriosin yang diproduksi oleh L. plantarum S34, disebut dengan plantarisin W (plnW), diamplifikasi dari plasmid dan dikloning menggunakan sistem vektor pGEM®-T Easy ke dalam Escherichia coli DH5?. Sekuens nukleotida plnW (± 405 pb) diidentifikasi sebagai protein integral membran. Lebih lanjut, plnW diekspresikan secara heterologus sebagai fusi protein dengan His(6)-tag tioredoksin menggunakan vektor ekspresi pET-32a(+) ke dalam E. coli BL21 (DE3) pLysS. Protein fusi rekombinan plnW terdapat dalam sitoplasma sel, tetapi selain fraksi terlarut terdapat juga fraksi tidak terlarut berupa badan inklusi. Purifikasi parsial dilakukan menggunakan kromatografi afinitas ligan Co2+ untuk fraksi terlarut dan metode elektroelusi gel poliakrilamid untuk fraksi tidak terlarut. Massa molekul berukuran kurang lebih 33 kDa terdeteksi berdasarkan pemisahan SDS-PAGE dan dikonfirmasi dengan Western blot sebagai protein fusi rekombinan plnW. Protein yang sudah terpurifikasi bermanfaat untuk mengetahui kaitan antara struktur dan fungsi bakteriosin.
Identification of Bioactive Compound from Microalga BTM 11 as Hepatitis C Virus RNA Helicase Inhibitor Mustopa, Apon Zaenal; Umami, Rifqiyah Nur; Putri, Prabawati Hyunita; Susilaningsih, Dwi; Farida, Hilda
JURNAL BIOLOGI INDONESIA Vol 11, No 2 (2015): JURNAL BIOLOGI INDONESIA
Publisher : Perhimpunan Biologi Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1133.514 KB) | DOI: 10.14203/jbi.v11i2.2198

Abstract

ABSTRACTHepatitis C virus (HCV) is the major causative agent of chronic liver disease. Recently, the inhibition of NS3 RNA helicase/ATPase activity is being explored as the specifically targeted antiviral therapy (STAT) against HCV infection. This study was aimed to elucidate potential candidates for anti-HCV therapy derived from Indonesian indigenous microalgae. The microalga designated as BTM 11 was isolated and cultured. Methanol extract of BTM 11 was screened as the opponent of purified HCV NS3 RNA helicase enzyme through colorimetric ATPase assay. Screening of chemical compound and fractionation by using gel filtration chromatography with eluent of methanol : chloroform (1:99) were conducted for identification and isolation of the bioactive compounds. The third fraction of fractionated sample showed a relatively strong ATPase inhibitory effect (81.23 ± 2.25 %) compared to the negative control. Further analysis of third fraction using thin layer chromatography (TLC) with eluent of chloroform : methanol (9:2) gave two spots with the Rf value of 0.8 and 0.37, respectively. In addition, high performance liquid chromatography (HPLC) analysis showed absorption peak with the highest abundance at the retention time of 12.483 and 16.617 minutes which absorbed at 266 and 230 nm wavelenght, respectively. According to those analyses, this study suggests that bioactive compounds derived from BTM 11 were classified as the groups of flavonoids and feasible as potential candidates for anti-HCV therapy through the inhibitory effect of NS3 RNA helicase/ATPase activity. Keywords: Hepatitis C Virus, NS3 RNA helicase, ATPase, Microalga, Flavonoids 
Karakteristik Fisikokimia Gelatin Tulang Ikan Patin (Pangasius sutchi) Hasil Ekstraksi Menggunakan Limbah Buah Nanas (Ananas comosus) Atma, Yoni; Ramdhani, Hisworo; Mustopa, Apon Zaenal; Pertiwi, Mega; Maisarah, Rizkia
Agritech Vol 38, No 1 (2018)
Publisher : Faculty of Agricultural Technology, Universitas Gadjah Mada, Yogyakarta, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (289.597 KB) | DOI: 10.22146/agritech.29821

Abstract

Fish bones are the most potential as an alternative source for gelatin production. Pangasius catfish bone is the one of the most promising source due to its high yield of gelatin. It has similar gel strength with the commercial gelatin and has a low ash content. The aim of this research was to determine the physical and chemical characteristics of Pangasius catfish bone gelatin extracted using pineapple waste. Pineapple waste has a low pH and contains citric acid. The research was conducted in two stages i.e. gelatin extraction and physicochemical characterization. Gelatin extraction was done by pre-treatment and main extraction. Pre-treatment was done by soaking the fish bone into pineapple waste for 32, 48, and 56 hours. The main extraction was carried out by soaking fish ossein in warm water for 5 hours at 75 °C.  The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the gelatin existence. Fish bone gelatin has molecular weight ranging from 100-150 kDa and > 225 kDa. The hydroxyproline content of gelatin was 10.9–16.3 mg/g. Gelatin of Pangasius catfish bone extracted with pineapple waste has gel strength of 64.83 g.bloom g, hardness of 4.96 N, cohesiveness of 0.88, springiness of 1.03 mm, gumminess of 4.36 N, and chewiness of 2.78 N. Viscosity and pH of gelatin solution obtained were 3.17 cP and 4.52 respectively. The proximate characteristics obtained were moisture 8.59%, ash 0.95%, crude protein 47.60% and fat 7.71%. Some physicochemical parameters of gelatin resemble commercial gelatin and other fish bone gelatin. ABSTRAKTulang ikan merupakan salah satu sumber alternatif untuk produksi gelatin yang sangat potensial. Gelatin dari tulang ikan patin yang diekstrak dengan asam diketahui memiliki hasil ekstraksi yang tertinggi, kekuatan gel yang menyerupai gelatin komersial dan kadar abu yang rendah. Penelitian ini dilakukan untuk mengetahui karakteristik fisik dan kimiawi gelatin tulang ikan patin yang diekstrak dengan limbah buah nanas. Limbah buah nanas diketahui mengandung asam sitrat cukup tinggi. Penelitian dilakukan dengan dua tahap yakni ekstraksi gelatin dan analisis karakteristik fisikokimianya. Ekstraksi gelatin dilakukan dengan dua tahap yakni pre-treatment dan ekstraksi utama. Pre-treatment dilakukan dengan perendaman tulang ikan dalam limbah buah nanas selama 32, 48, dan 56 jam. Ekstraksi utama dilakukan dengan perendaman dalam air hangat selama 5 jam suhu 75 °C. Analisis keberadaan gelatin dengan metode SDS-PAGE diketahui gelatin berada pada kisaran berat molekul 100–150 kDa dan > 225 kDa. Kadar hidroksiprolin gelatin yakni 10,9–16,3 mg/g. Gelatin tulang ikan patin yang diekstrak dengan limbah buah nanas memiliki kekuatan gel 64,83 g.bloom, kekerasan (hardness) 4,96 g, kohesivitas (cohesiveness) 0,88, elastisitas (springiness) 1,03, kekenyalan (gumminess) 4,36, daya kunyah (chewiness) 2,78. Viskositas dan pH larutan gelatin yang diperoleh masing-masing 3,17 cP dan 4,52. Karakteristik kimiawi gelatin yang diperoleh antara lain meliputi kadar air 8,59%, abu 0,95%, protein kasar 47,60% dan lemak 7,71%. Beberapa karakteristik fisikokimia gelatin tulang ikan pada penelitian ini dapat dibandingkan dengan gelatin komersial dan gelatin tulang ikan yang lain.
Enterococcus faecium 1.15 Isolated from Bakasam Showed Milk Clotting Activity Putranto, Wendry Setiyadi; Suradi, Kusmajadi; Chairunnisa, Hartati; Mustopa, Apon Zaenal; Giriwono, Puspo Edi; Kusumaningrum, Harsi Dewantari; Suhartono, Maggy Thenawidjaja
ANNALES BOGORIENSES Vol 21, No 1 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (499.913 KB) | DOI: 10.14203/ab.v21i1.293

Abstract

The Lactic Acid Bacteria with Milk Clotting Activity (MCA) were isolated from Bakasam, an Indonesian traditional fermented meat. The isolate screening was carried out using modified method of Skim Milk Agar and Milk Clotting Activity Test, and the isolate was then identified using 16S rRNA. We found 4 isolates that showed MCA of 18-20 SU/ml. Identification using 16S rRNA indicated that the isolate ALG.1.15 was 99% (FR3-F primer) and 99% (FR3-R primer) identic with Enterococcus faecium. The isolate potentially produced renin-like protease to subtitute renin from veal.