Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily

Cloning, Expression, and In Silico Analysis of Class IV Poly-(R)-3-hydroxybutyrate Genes from New Strain of Bacillus thuringiensis TH-01

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

Cloning, sequencing, and identification of rhd ?-subunit gene of haloaromatic dehalogenase terminal oxygenase from Pseudomonas aeruginosa local strain

Haloaromatic is one of organohalogen pollutants found in the environment. These compounds are toxic, persistent, carcinogenic, and mutagenic. Accumulations of these compounds in the environment may cause serious diseases in organisms, including humans. Previous research results showed that breaking the bond of halogen atom from the aromatic ring could eliminate the toxicity of these compounds. Haloaromatic dehalogenase is an enzyme that possesses an ability to cut the halogen atom from the aromatic ring. In this research, a rhd α-subunit gene of haloaromatic dehalogenase terminal oxygenase has been isolated and sequenced. The gene was isolated by PCR approach from Pseudomonas aeruginosa local strain, which is a Gramnegative bacterium found in soil as a pioneer growth bacterium. The sequence obtained indicated that rhd α-subunit gene is 1290 bp in size. The RHD α-subunit deduced amino acid has Cys98-X1-His100-X17-Cys118-X2-His121 and His217-X4- His222-X149-Asp372 motifs, two important motifs in aromatic ring hydroxylating dioxygenase family, which is a large family of multi subunit and multifunctional proteins. One of its functions is in the breaking of halogen element from the aromatic carbon ring. Omori classification showed that RHD α-subunit is classified as Group II of aromatic ring hydroxylating dioxygenase family, which includes haloaromatic dehalogenases. The result of 3D structure prediction also indicated that this protein belongs to this large family with 54 percentile Mol Probity score

Mutation on Bacillus BAC4 Using Acridine orange as an Effort for Increasing Antibiotic Production = Mutasi pada Bacillus subtilis BAC4 dengan larutan akridin oranye sebagai upaya peningkatan produksi antibiotika

ABSTRACT The efforts to get a new antibiotic require to be done continuously, because infection diseases still become the main health problems in Indonesia. A new local strain of Bacillus subtilis BAC4 has been known producing an antibiotic that inhibites Serratia marcescens A TCC 27117 growth. Nevertheless, the optimum conditions have not been studied seriously. The objective of this research was to conduct mutation on B. subtilis BAC4 in order to obtain a mutant cell that overproducts In producing antibiotic. The mutation process was performed by using acridine orange of 1 g.C\u27 randomly at various volumes. The production of antibiotic was conducted using batch fermentation and antibiotic assay was performed with Elgar absorption method using S. marcescens ATCC 27117 as bacteria assay. Research result provided a B. subtilis M10 mutant with overproduction of antibiotic. Characterization of B. subtilis M10 mutant showed that the mutant cell has size of (0.5-1.0) pm x (1.85-2.5) pmspore has the form of ellipse with thick wavy wall, positive reaction for catalase, and fO(111ing acid from glucose and xylose Keywords: mutant, Bacillus, acridin, and antibiotics

Expression of haloacid dehalogenase gene and its molecular protein characterization from Klebsiella pneumoniae ITB1

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

Identifikasi Senyawa Alkaloid dari Akar Piper sarmentosum Roxb. Ex Hunter dan Uji Aktivitasnya terhadap Jamur Candida albicans

Piper sarmentosum Roxb. Ex Hunter or “Sirih duduk” has long been used for traditional medicine to cure various diseases, such as fungus infections. The investigation of the bioactive compounds of P. sarmentosum roots has not been carried out. This research was aimed to isolate the bioactive compounds from P. sarmentosum roots. The results showed that methanol extracts of P. sarmentosum roots have an activity on Candida albicans. The separation a bioactive compounds from methanol extracts of P. sarmentosum roots was performed by column chromatography, thin layer chromatography and recrystalizations. The identifications of the bioactive compounds were carried out using ultra violet spectrometry, infrared spectrometry, gas chromatography-mass spectrometry and nuclear magnetic resonance spectrometry. The results indicated that from methanol extracts, an alkaloid compound of piperoylpyrol derivative was 5-hydroxy-5- (3,4-methylenedioxyphenyl)-2-pentenoyl pyrol, could be purely isolated. Examination of bioactivity at concentration 0.10-2.50 mg/ml showed that this compound had an activity on C. albicans

Cloning of acetyl-CoA acetyltransferase gene from Halomonas elongata BK-AG18 and in silico analysis of its gene product

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

GAMBARAN TINGKAT PENGETAHUAN REMAJA TENTANG KEPUTIHAN

Keputihan adalah keluarnya cairan vagina selain darah. Kurangnya pengetahuan remaja putri tentang informasi yang tepat tentang kesehatan organ reproduksi dapat menimbulkan kurangnya perhatian terhadap kesehatan organ reproduksi, seperti keputihan. Dampak dari keputihan yang terlambat atau tidak dipbat dapat berakibat buruk bagi kehidupan seorang wanita, seperti terjadinya infertil, endometritis, radang panggul dan salpingitis. Tujuan penelitian ini untuk mengetahui gambaran tingkat pengetahuan remaja tentang keputihan. Penelitian ini menggunakan metode literature review. Populasi dalam penelitian ini adalah remaja putri. Hasil penelitian literatur review ini menunjukkan bahwa penelitian rata-rata pengetahuan remaja tentang keputihan sangat kurang, terdapat faktorfaktor yang mempengaruhi tingkat pengetahuan remaja yaitu faktor pendidikan formal, usia, ekonomi, informasi. Bidan diharapkan dapat memberikan sosialisasi seperti penyuluhan kepada remaja ke sekolah terkait dengan tingkat pengetahuan mereka tentang menjaga kesehatan reproduksi sepertikeputihan