Isolation and Characterization of Organic-Solvent Stable Protease Isolated by Pseudomonas stutzeri BK AB-12

AbstractIn this study, proteases have been isolated from halophilic bacteria, Pseudomonas stutzeri BK AB-12. The bacteria produced proteases as exhibited by the appearance of clear zones resulted from casein hydrolysis. Based on the protease specific activity, 70−80% ammonium fraction exhibited the highest activity. Protease in this fraction has highest activity at pH 8.0 and 55°C and its activity of protease was enhanced by the addition of several metal ions. The addition of Fe3+ ion also resulted in a shifting of the optimum pH from 8 to 9 and the optimum temperature from 55 to 60°C. Protease of P. stutzeri BK AB-12 in the fraction of 70−80% is not likely metallo-, cysteine-, or serine-protease because it is not inhibited by EDTA, β-mercaptoethanol, and PMSF. Proteases of this fraction was sensitive to ionic strength, where the highest activity was observed at concentrations at NaCl concentrations of 2.5M. Beside influenced by the ionic strength, protease activity was also sensitive to solvent polarity. The protease wasstable in the presence of different organic solvents, which enables its potential use for the synthesis of peptides

Estimating Factors Determining Emulsification Capability of Surfactant-Like Peptide with Coarse-Grained Molecular Dynamics Simulation

The ability of surfactant-like peptides to emulsify oil has become the main focus of our current study. We predicted the ability of a series of surfactant-like peptides (G6D, A6D, M6D, F6D, L6D, V6D, and I6D) to emulsify decane molecules using coarse-grained molecular dynamics simulations. A 1-μs simulation of each peptide was carried out at 298 K and 1 atm using MARTINI force field. Simulation system was constructed to consist of 100 peptide molecules, 20 decane molecules, water, antifreeze particles and neutralizing ions in a random configuration. Out of seven tested peptides, M6D, F6D, L6D, V6D, and I6D were able to form emulsion while G6D and A6D self-assembled to order b-strands. A higher hydropathy index of amino acids constituting the hydrophobic tail renders the formation of an emulsion by peptides more likely. By calculating contact number between peptides and decanes, we found that emulsion stability and geometry depends on the structure of amino acids constituting the hydrophobic tail. Analysis of simulation trajectory revealed that emulsions are formed by small nucleation following by fusion to form a bigger emulsion. This study reveals the underlying principle at the molecular level of surfactant peptide ability to form an emulsion with hydrophobic molecules

Novel Archaeal DNA Polymerase B from Domas Hot Spring West Java

Nine novel archaeal DNA polymerase genes from Domas Hot Spring, West Java have been cloned directly through the natural sample. The characterization of the genes showed that the genes are high homology to the DNA polymerase B of Crenarhaea phyla. Phylogenetic analysis of the amino acid sequences showed that the enzymes are grouped in a new branch from the other Crenarchaea’s DNA Polymerase B. 3D structure analysis of the enzymes show that the structures are closed to the structure of DNA Polymerase B1 from Sulfolobus solfataricus. The nine structures of the enzymes could be grouped into four different structures

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

Screening and Characterization of Levan Secreted by Halophilic Bacterium of Halomonas and Chromohalobacter Genuses Originated from Bledug Kuwu Mud Crater

AbstractFive bacterial isolates from the genus of Halomonas and Chromohalobacter isolated from Bledug Kuwu mud crater, located in mainland Purwodadi-Grobogan, Central Java, Indonesia, have been assayed for levan production. Initial screening was conducted on modified Belgith medium using sucrose as the major carbon source, in which the colonies of levan-producing bacteria will have a slimy mucoid appearance when grown on this medium. The screening results showed only one positive bacterial species, which was Chromohalobacter japonicus BK AB18 that identified as potential levan producer. Thermal stability of the isolated levan has been charachterized by TGA, which gave approximate decomposition temperature about 211 oC. The structure of the levan has been elucidated by FTIR and NMR spectroscopies. FTIR spectrum of the isolated levan displayed high similarity to that of levan isolated from Bacillus methylotrophicus. The chemical shifts of carbon and proton NMR spectra of the isolated levan also exhibited high similiarity to those of levan isolated from Pseudomonas fluorescens and Zymonas mobils

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

Produksi dan Optimasi Biosurfaktan dari Bakteri Halofilik Chromohalobacter japonicus BK-AB18

Perkembangan teknologi bioproses telah mendorong pendekatan ke arah produk surfaktan biologi (biosurfaktan) yang ramah lingkungan. Penelitian ini difokuskan pada optimasi produksi biosurfaktan dengan menggunakan variasi sumber karbon dan nitrogen. Tahap awal penelitian ini dilakukan dengan menguji potensi bakteri Chromohalobacter japonicus BK-AB18 penghasil biosurfaktan dengan  menggunakan metode uji hemolisis dimana hasil menunjukkan aktivitas hemolisis yang tinggi dengan  ukuran zona bening 3 cm pada media agar darah. Selanjutnya dilakukan produksi pada medium optimal dengan menambahkan 2% variasi sumber karbon terdiri dari minyak zaitun, minyak jagung, minyak kelapa sawit, minyak kacang kedelai dan minyak bunga matahari serta 0,3 % variasi nitrogen terdiri dari urea, NaNO3, NH4Cl, NH2(SO)4 and KNO3. Hasil penelitian menunjukkan produksi biosurfaktan dari Chromohalobacter japonicus BK-AB18 meningkat secara signifikan dengan menggunakan minyak zaitun sebagai sumber karbon dan urea sebagai sumber nitrogen dengan hasil penyebaran minyak 4,8 cm serta aktivitas biosurfaktan dengan nilai tegangan permukaan sebesar 34 dyne/cm, dan hasil uji emulsifikasi sebesar 76%

Docking and Molecular Dynamics Simulation of Carbonic Anhydrase II Inhibitors from Phenolic and Flavonoid Group

AbstractCarbonic Anhydrase II (CAII) has role in pH regulation, water transport and hydration of CO2. In addition, CAII is also related to many diseases, including glaucoma, tumours, epilepsy, diabetes and osteopetrosis. Various inhibitors for CAII have been developed and commercialized as a drug. Recent development of CAII inhibitors drive the invention of novel inhibitors based on natural product structures and their derivatives. This research aim to screen potential inhibitors from phenolic and flavonoid groups by in silico approach. The screening of natural products compounds was performed by a molecular docking method. The best ligand derived from the molecular docking selection was further refined with a molecular dynamics simulation and the resulted structure was used to evaluate the stability of CAII-ligand complex. By using the upper mentioned procedures, fisetin (Fic) and 6-(3,4-dihydroxyphenyl)-5,6,7,8-tetrahydronaphthalene-1,3,7-triol (Afr3) were strongly suggested to be a potent inhibitor for CAII

Effect of calcium ions to the activity and stability of extracellular lipase produced by moderate halophilic bacteria Halomonas meridiana BK-AB4

This study reported the isolation and biochemical studies of extracellular lipase produced by a moderate halophilic bacterium, Halomonas meridiana BK-AB4. The estimated molecular weight of the lipase BK-AB4 is about 50 kDa. The study of metal ions effect to the enzyme activity revealed the strong dependency towards the presence of Ca2+ ion. Further studies showed that the optimum pH of the enzyme was similar in the presence and the absence of Ca2+ ions. However, the presence of Ca2+ ions could improve the enzyme thermal stability as indicated by the shift of its optimum temperature from 45°C - 50°C. The addition of Ca2+ ions was also enhancing residual activity towards various inhibitors, such as SDS and PMSF up to 55% and 68%, respectively. Another interesting effect of the addition of Ca2+ ions were noted when various organic solvents were added to the enzyme solution in the ratio of 1:1. The activity of the enzyme increased up to 122% and 126% relative to the control when methanol and n-butanol were added, respectively. This study thus showed that the addition of Ca2+ ion not only improved the catalytic activity of lipase BK-AB4 but also made the enzyme more tolerable against various perturbations

Determination of activities of human carbonic anhydrase II inhibitors from curcumin analogs

Purpose: To evaluate the activities of new curcumin analogs as carbonic anhydrase II (CA-II) inhibitor.Methods: Carbonic anhydrase II (CA-II) inhibition was determined by each ligand capability to inhibit the esterase activity of CA-II using 4-NPA as a substrate in 96-well plates. Dimethyl sulfoxide was used to dissolve each curcumin analog compound, and then diluted with biological buffer. They were then mixed with CA-II solution and to start the reaction, 4-NPA was added. Hydrolysis of the substrate was evaluated at 405 nm after incubation for 2 h at 25 °C. The IC50 value of compounds with inhibitory activity higher than 40 % was then evaluated. Molecular docking was also used to predict enzymeinhibitor interaction.Results: Eight new curcumin analogs were potent to inhibit CA-II activity with IC50 values ranging from 7.92 ± 0.54 to 72.31 ± 2.21 μmol; the lowest value was exhibited by (3E,5E)-3,5-bis[(2- hydroxyphenyl)methylidene]piperidin-4-one (a1). Molecular docking analysis revealed that this molecule formed hydrogen bonds with Thr199, Thr200 and Gln92 at the active site of CA-II.Conclusion: These curcumin analogs have inhibitory potential against CA-II; (3E, 5E)-3,5-bis[(2- hydroxyphenyl)methylidene]piperidin-4-one (a1) has the highest inhibitory activity and may be useful in the development of CA-II inhibitors for glaucoma treatment.Keywords: Carbonic anhydrase II inhibitor, Curcumin analogs, Molecular dockin