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Purifikasi Protein Fusi MBP-Mga Streptococcus pyogenes Hasil Ekspresi Heterolog di Escherichia coli Muhaimin Muhaimin; Oei Ban Liang; Enny Ratnaningsih; Endang Purwantini; Debbie Sofie Retnoningrum
Jurnal Matematika & Sains Vol 10, No 1 (2005)
Publisher : Institut Teknologi Bandung

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Abstract

Optimization of purification process of Streptococcus pyogenes’s MBP-Mga fusion protein from Escherichia coli pMALMga carrying mga49 gene had been carried out. The purification was conducted by using affinity chromatography column with amylose ligands. SDS-PAGE analysis gave a band at 104 kDa showing a pure MBP-Mga fusion protein.
Optimasi Proses Overproduksi, Pemurnian dan Karakterisasi Protein Mga Sebagai Molekul Target Untuk Pencegahan Infeksi oleh Streptococcus Pyogenes Muhaimin Muhaimin; Oei Ban Liang; Enny Ratnaningsih; Endang Purwantini; Debbie Soefie Retnoningrum
Jurnal Matematika & Sains Vol 8, No 3 (2003)
Publisher : Institut Teknologi Bandung

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Abstract

Optimization of overproduction, purification and characterization of Streptococcus pyogenes’s Mga recombinant protein from Escherichia coli pMALMga carrying mga49 gene had been carried out. The overproduction optimization was conducted by culturing the Escherichia coli pMALMga of OD600 = 0,7 in LB medium with or without glucose, and induced by 0,3 mM IPTG for a certain period of time. The protein produced was MBP-Mga fusion protein with a molecular weight of about 104 kDa. The purification was conducted by affinity chromatography using amylose ligands at pH 7,4 and continued by SDS-PAGE. One band identified at 104 kDa showed that the MBP-Mga fusion protein was pure enough for further analysis. The MBP-Mga protein was then injected into rabbit to produce polyclonal antibodies. The antibody development was monitored by dot blot and Western blot. The antibodies produced gave a positive reaction to MBP-Mga fusion protein. Adsorption and precipitation of the polyclonal antibodies which gave a positive reaction to MBP protein was done by adding pure MBP protein (56 kDa) produced by Escherichia coli pMALc2, and purified by affinity chromatography using amylose ligands at pH 7,4. The results showed that the antibodies gave a positive reaction to Mga recombinant protein and a negative reaction to MBP recombinant protein.
IDENTIFIKASI DAN ISOLASI HALOALKANA DEHALOGENASE DARI PSEUDOMONAS AERUGINOSA STRAIN LOKAL Nora, Adri; Ratnaningsih, Enny; Natalia, Dessy
Indonesian Journal of Biotechnology and Biodiversity Vol 1, No 1 (2017): Indonesian Journal of Biotechnology and Biodiversity
Publisher : Indonesian Journal of Biotechnology and Biodiversity

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Abstract

AbstrakSenyawa organohalida banyak digunakan dalam industri sebagai pestisida dan aditif untuk bensin. Salah satu senyawa organohalida yang banyak diproduksi adalah 1,2-dikloroetana (DCE). Namun, senyawa organohalida merupakan polutan yang dapat membahayakan lingkungan karena senyawa ini sulit terdegradasi dan bersifat karsinogenik. Beberapa bakteri diketahui mampu mendegradasi DCE seperti bakteri dari genus Pseudomonas, Bradyrizhobium, dan Xantobacter. Bakteri-bakteri tersebut menghasilkan haloalkana dehalogenase yang mampu mengkatalisis pemutusan ikatan antara karbon dengan halogen. Pada DCE katalisis dehalogenase menghasilkan 2-kloroetanol, ion halida, dan  proton. Dalam penelitian ini dilakukan identifikasi dan isolasi haloalkana dehalogeanse dari Pseudomonas aeruginosa strain lokal. Pseudomonas aeruginosa diidentifikasi dengan PCR16S ribosomal DNA menggunakan Unibi (5’-GGT TAC (GC) TTG TTA CGA CTT-3) sebagai primer maju dan BactF-1 (5’-AGA GTT TGA TCA CTG GCT CAG-3’) sebagai primer mundur. Pseudomonas aeruginosa dapat tumbuh dalam medium Luria Bertani dan minimal medium yang mengandung DCE 1─10mM. Haloalkana dehalogenase yang dihasilkan merupakan enzim intrasel dengan aktivitas spesifik 8,809 unit/mg protein (unit = µmol Cl- / menit). Isolasi dan fraksinasi haloalkana dehalogenase menghasilkan aktivitas spesifik menjadi 32,108 unit/mg protein (naik 3,5 kali dibandingkan ekstrak kasarnya). Jika dibandingkan dengan Pseudomonas aeruginosa OK1, Pseudomonas aeruginosa strain lokal ini lebih berpotensi untuk digunakan dalam bioremediasi karena Pseudomonas aeruginosa strain lokal memiliki aktivitas spesifik yang lebih besar dan kemampuan tumbuh yang lebih cepat dibandingkan OK1. Kata Kunci : 1,2-dikloroetana, Pseudomonas aeruginosa, haloalkana dehalogenase
Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1 Ratnaningsih, Enny; Ade, Rachmad; Putri, Rindia Maharani; Idris, Idris
International Journal of Renewable Energy Development Vol 10, No 4 (2021): November 2021
Publisher : Center of Biomass & Renewable Energy, Diponegoro University

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14710/ijred.2021.38887

Abstract

In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37ºC with shaking, 2 hours IPTG induction at 30ºC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation
IDENTIFIKASI DAN ISOLASI HALOALKANA DEHALOGENASE DARI PSEUDOMONAS AERUGINOSA STRAIN LOKAL Nora, Adri; Ratnaningsih, Enny; Natalia, Dessy
Indonesian Journal of Biotechnology and Biodiversity Vol 1, No 1 (2017): Indonesian Journal of Biotechnology and Biodiversity
Publisher : Universitas Esa Unggul

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Abstract

AbstrakSenyawa organohalida banyak digunakan dalam industri sebagai pestisida dan aditif untuk bensin. Salah satu senyawa organohalida yang banyak diproduksi adalah 1,2-dikloroetana (DCE). Namun, senyawa organohalida merupakan polutan yang dapat membahayakan lingkungan karena senyawa ini sulit terdegradasi dan bersifat karsinogenik. Beberapa bakteri diketahui mampu mendegradasi DCE seperti bakteri dari genus Pseudomonas, Bradyrizhobium, dan Xantobacter. Bakteri-bakteri tersebut menghasilkan haloalkana dehalogenase yang mampu mengkatalisis pemutusan ikatan antara karbon dengan halogen. Pada DCE katalisis dehalogenase menghasilkan 2-kloroetanol, ion halida, dan  proton. Dalam penelitian ini dilakukan identifikasi dan isolasi haloalkana dehalogeanse dari Pseudomonas aeruginosa strain lokal. Pseudomonas aeruginosa diidentifikasi dengan PCR16S ribosomal DNA menggunakan Unibi (5’-GGT TAC (GC) TTG TTA CGA CTT-3) sebagai primer maju dan BactF-1 (5’-AGA GTT TGA TCA CTG GCT CAG-3’) sebagai primer mundur. Pseudomonas aeruginosa dapat tumbuh dalam medium Luria Bertani dan minimal medium yang mengandung DCE 1─10mM. Haloalkana dehalogenase yang dihasilkan merupakan enzim intrasel dengan aktivitas spesifik 8,809 unit/mg protein (unit = µmol Cl- / menit). Isolasi dan fraksinasi haloalkana dehalogenase menghasilkan aktivitas spesifik menjadi 32,108 unit/mg protein (naik 3,5 kali dibandingkan ekstrak kasarnya). Jika dibandingkan dengan Pseudomonas aeruginosa OK1, Pseudomonas aeruginosa strain lokal ini lebih berpotensi untuk digunakan dalam bioremediasi karena Pseudomonas aeruginosa strain lokal memiliki aktivitas spesifik yang lebih besar dan kemampuan tumbuh yang lebih cepat dibandingkan OK1. Kata Kunci : 1,2-dikloroetana, Pseudomonas aeruginosa, haloalkana dehalogenase
Characterization of Extracellular Penicilin G Acylase Produced by A New Local Strain of Bacillus subtilis BAC4 SUPARTONO SUPARTONO; ENNY RATNANINGSIH; SADIJAH ACHMAD; OEI BAN LIANG
HAYATI Journal of Biosciences Vol. 15 No. 2 (2008): June 2008
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (130.423 KB) | DOI: 10.4308/hjb.15.2.71

Abstract

Penicillin G acylase (PGA) which catalyses penicillin G hydrolysis reaction is a key enzyme for the industrial production of penicilin G derivatives used in therapeutics. A new local strain of Bacillus subtilis BAC4 was found capable of producing extracellular PGA. However, characteristics of this extracellular PGA are not known. The goal of this research was to characterize the extracellular PGA produced by B. subtilis BAC4. Enzyme production was carried out by batch fermentation, followed by enzyme purification and characterization of the PGA. The PGA activity was determined by the Kornfeld method, with optimal activity for hydrolysing penicillin G observed at 43 oC and pH 8.5. The activation energy of penicillin G hydrolysis by the PGA of B. subtilis BAC4 was determined as 4.9 kcal.mol-1 and Vmax and Km values were found to be 0.7 µmole.min-1.mg-1 and 3.5 mM respectively. PGA catalytic activity was competitively inhibited by phenylacetic acid with an inhibition constant, Ki(PAA) , of 347.2 mM. It was concluded that the extracellular PGA of B. subtilis BAC4 can hydrolyse penicillin G efficiently. Key words: PGA, extracellular, Bacillus, local
Cloning, Expression, and In Silico Analysis of Class IV Poly-(R)-3-hydroxybutyrate Genes from New Strain of Bacillus thuringiensis TH-01 Zuhdina Sabiqoh; Rukman Hertadi; Enny Ratnaningsih
HAYATI Journal of Biosciences Vol. 29 No. 3 (2022): May 2022
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.29.3.310-319

Abstract

Poly-(R)-3-hydroxybutyrate (PHB) is a bioplastic derivative of polyhydroxyalkanoate (PHA) which can be synthesized by bacteria under certain growth conditions. Previous study has reported a new strain of Bacillus thuringiensis TH-01 isolated from thermite, which found to accumulate PHB. This research aimed to clone PHB biosynthesis genes from B. thuringiensis TH-01 and study its expression as well as predict the tertiary structure of the enzymes. The clone of phaA gene, which encodes PhaA, was obtained as 1182 bp. On the other hand, 2546 bp clone of phaRBC gene cluster was obtained to consist of 744 bp phaB, 1086 bp phaC, and 483 bp phaR, encoding respective PhaB, PhaC, and PhaR proteins. In silico analysis indicated that PhaA, PhaB, PhaC, and PhaR, revealed to have 393, 247, 361, and 160 amino acid, respectively. The predicted model of PhaA, PhaB, and PhaC showed dominant structure of α/β folding motif, while PhaR was dominated by a helix-loop-helix motif. The catalytic residues of PhaA were Cys88, His349, and Cys379, whereas the catalytic residues of PhaB were Ser142, Tyr155, and Lys159. These catalytic residues were identical to those residues obtained in other PHB biosynthetic enzymes reported elsewhere, confirming that our clones were of PHB biosynthetic genes.
Membrane Oxygenator for Extracorporeal Blood Oxygenation Enny Ratnaningsih; Putu T.P. Aryanti; Nurul F. Himma; Anita K. Wardani; K. Khoiruddin; Grandprix T.M. Kadja; Nicholaus Prasetya; I Gede Wenten
Journal of Engineering and Technological Sciences Vol. 53 No. 5 (2021)
Publisher : Institute for Research and Community Services, Institut Teknologi Bandung

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.5614/j.eng.technol.sci.2021.53.5.2

Abstract

Extracorporeal blood oxygenation has become an alternative to supply O2 and remove CO2 from the bloodstream, especially when mechanical ventilation provides insufficient oxygenation. The use of a membrane oxygenator offers the advantage of lower airway pressure than a mechanical ventilator to deliver oxygen to the patient’s blood. However, research and development are still needed to find appropriate membrane materials, module configuration, and to optimize hydrodynamic conditions for achieving high efficient gas transfer and excellent biocompatibility of the membrane oxygenator. This review aims to provide a comprehensive description of the basic principle of the membrane oxygenator and its development. It also discusses the role and challenges in the use of membrane oxygenators for extracorporeal oxygenation on respiratory and cardiac failure patients.
Expression of haloacid dehalogenase gene and its molecular protein characterization from Klebsiella pneumoniae ITB1 Ridani Rino Anggoro; Enny Ratnaningsih
Indonesian Journal of Biotechnology Vol 22, No 1 (2017)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (603.006 KB) | DOI: 10.22146/ijbiotech.26004

Abstract

Organohalogen compounds are widely used industrially and agriculturally, as well as in households as flame retardants and refrigerants. However, these compounds can become significant pollutants through their accidental or deliberate release into the environment in large quantities. Dehalogenase is an enzyme with the potential to be used in the removal of organohalogen contaminants. A previous study successfully subcloned a 690 bp of haloacid dehalogenase gene (hakp1) from Klebsiella pneumoniae ITB1 into a pET-30a(+) expression system to achieve high enzyme productivity. IPTG was used as an inducer to express a pET-hakp1 recombinant clone in Escherichia coli BL21 (DE3). The molecular mass of the haloacid dehalogenase Hakp1 protein was 30 kDa as determined by SDS-PAGE. Zymogram analysis showed that this recombinant protein has dehalogenase activity as shown by the formation of AgCl white precipitate. A quantitative assay of haloacid dehalogenase Hakp1 gave a specific activity of 84.29 U/mg with the optimum temperature of 40°C at pH 9. Predicted three-dimensional structure of Hakp1 showed α/β motif folding which comprised of cap and core domain. The predicted active sites of Hakp1 were Asp8, Glu10, Leu22, Phe23, Trp90, Ser125, Ser126, Lys159, and Asp184 with Asp8, Glu10, Ser126, and Lys159 act as binding residue. This recombinant haloacid dehalogenase clone provides an alternative agent for effective bioremediation of organohalogen pollutants.
Cloning of acetyl-CoA acetyltransferase gene from Halomonas elongata BK-AG18 and in silico analysis of its gene product Ni Putu Yuliastri; Enny Ratnaningsih; Rukman Hertadi
Indonesian Journal of Biotechnology Vol 22, No 1 (2017)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (481.333 KB) | DOI: 10.22146/ijbiotech.27235

Abstract

Polyhydroxybutyrate (PHB) is a biodegradable polymer that can be used as a substitute for petrochemical plastics. Bacteria accumulate PHB in their cells as carbon and energy reserves because of unbalanced growth conditions.  This study aimed to amplify phbA from the chromosomal DNA of Halomonas elongata BK-AG18, a PHB-producing bacterium that was previously isolated from the Bledug Kuwu mud crater of Central Java, Indonesia. The obtained phbA amplicon was 1176 bp. This fragment was cloned into a pGEM-T Easy cloning vector and used to transform Eschericia coli TOP10. The recombinant colonies were selected using blue-white screening, confirmed by size screening, reconfirmed by re-PCR, and sequenced. When putative phbA sequences were aligned with H. elongata DSM2581 chromosome using BLASTN, this sequence showed 99% identity. The deduced amino acid sequences of this clone showed 100% identity to PhbA of  H. elongata DSM2581, suggesting that the obtained cloned fragment is a  phbA  gene. The 3D structure predicted by I-TASSER showed that PhbA of H. elongata  BK-AG18 had a high similarity to the acetyl CoA acetyltransferase structure of  Ralstonia eutropha H16. PhbA of H. elongata BK-AG18 possesses three catalytic residues, namely Cys88, His348, and Cys378.