Further analysis of burkholderia pseudomallei mf2 and identification of putative dehalogenase gene by pcr

Halogenated organic compounds are extensively and widely used as pesticides, herbicides, and antibiotics that contribute to the pollution. This research was aimed to further analyze and characterize a bacterium that has the ability to utilize 2,2-dichloropropionic acid (2,2-DCP) as a model to study dehalogenase enzyme production. Microscopic observation, biochemical tests and PCR technique were carried out in order to characterize the isolated bacterium. Strain MF2 showed its ability to grow on 10 mM 2,2-DCP liquid minimal medium with doubling time of 13 h with maximum chloride ion released of 19.8 μmolCl–/mL. The 16S rDNA analysis suggested that strain MF2 belongs to the genus Burkholderia. This was supported by the microscopic observation and biochemical tests. Dehalogenase gene was observed when using only primers dehIfor1 and dehIrev2 derived from group I deh PCR primer sequences, whereas no amplification using dhlB-314-forward and dhlB-637-reverse (group II dehalogenase) and haloacetate dehalogenase (H2-1157-forward and H2-1662-reverse) PCR primer sequences. The results suggested that, possibly, dehalogenase from MF2 was related to group I deh. In conclusion, strain MF2 showed the ability to utilize 2,2-DCP as sole source of carbon and energy. Further analysis revealed the MF2 strain consisted of dehalogenase gene that could be used for degradation of man-made halogenated compounds present in the environment. Using existing dehalogenase PCR primers, it was possible to amplify the dehalogenase genes sequence

In silico molecular analysis of novel L-specific dehalogenase from Rhizobium sp. RC1

Aims: This study presents the first structural model and proposed the identity of four important key amino acid residues, Asp13, Arg51, Ser131 and Asp207 for the stereospecific haloalkanoic acid dehalogenase from Rhizobium sp. RC1. Methodology and results: The enzyme was built using a homology modeling technique; the structure of crystallized LDEX YL from Pseudomonas sp. strain YL as a template. Model validation was performed using PROCHECK to generate the Ramachandran plot. The results showed 80.4% of its residues were located in the most favoured regions suggested that the model is acceptable. Molecular dynamics simulation of the model protein was performed in water for 10 nanoseconds in which Na+ was added to neutralize the negative charge and achieved energy minimization. The energy value and RMSD fluctuation of Ca backbone of the model were computed and confirmed the stability of the model protein. Conclusion, significance and impact of study: In silico or computationally based function prediction is important to complement with future empirical approaches. L-haloacid dehalogenase (DehL), previously isolated from Rhizobium sp. RC1 was known to degrade halogenated environmental pollutants. However, its structure and functions are still unknown. This structural information of DehL provides insights for future work in the rational design of stereospecific haloalkanoic acid dehalogenases

Genomic analysis of functional haloacid-degrading gene of bacillus megaterium strain bhs1 isolated from blue lake (mavi göl, turkey)

Purpose: Bacillus megaterium strain BHS1, isolated from an alkaline water sample taken from Mavi Golu (Blue Lake, Turkey), can grow on minimal medium containing 2,2-dichloropropionic acid. We characterized this bacterium at the genomic level. Methods: The HiSeq platform was used to carry out genome sequencing, de novo assembly, and scaffolding with strain BHS1. Next, genome data were analyzed to demarcate DNA regions containing protein-coding genes and determine the function of certain BHS1 genes. Finally, results from a colorimetric chloride ion-release assay demonstrated that strain BHS1 produces dehalogenase. Results: De novo assembly of the BHS1 genomic sequence revealed a genome size of similar to 5.37 Mb with an average G+C content of 38%. The predicted nuclear genome harbors 5509 protein-coding genes, 1353 tRNA genes, 67 rRNA genes, and 6 non-coding (mRNA) genes. Genomic mapping of strain BHS1 revealed its amenability to synthesize two families of dehalogenases (Cof-type haloacid dehalogenase IIB family hydrolase and haloacid dehalogenase type II), suggesting that these enzymes can participate in the catabolism of halogenated organic acids. The mapping identified seven Na+/H+ antiporter subunits that are vital for adaptation of the bacterium to an alkaline environment. Apart from a pairwise analysis to the well-established L-2-haloacid dehalogenases, whole-cell analysis strongly suggested that the haloacid dehalogenase type II might act stereospecifically on L-2-chloropropionic acid, D,L-2-chloropropionic acid, and 2,2-dichloropropionic acid. Whole-cell studies confirmed the utilization of these three substrates and the gene's role in dehalogenation. Conclusions: To our knowledge, this is the first report of the full genome sequence for strain BHS1, which enabled the characterization of selected genes having specific metabolic activities and their roles in the biodegradation of halogenated compounds

Isolation and characterization of a novel 2, 2-DCP-degrading Achromobacter sp. M×2 isolated from contaminated seawater of Desaru Beach, Southern Malaysia

Halogenated Organic Compounds (HOC) are major pollutants in the environment due to their wide usage as biocides in the agricultural industry. Hence, it is important that safe methods to eliminating such compounds are developed. Consequently, the goal of this study is to isolate and identify bacterial species capable of utilizing 2,2 Dichloropropionic acid (2,2-DCP). Using enrichment culture, a bacterium strain designated as M×2 was isolated from contaminated seawater located at desaru beach, Malaysia. The isolate grew best at 20 mM of 2,2-DCP minimal media with doubling time of 13.9 h. More than 0.59 mmol/L chloride ions were detected in the growth medium. Morphological and biochemical characteristics of M×2 were closely matched to those of Achromobacter sp. and the 16S rDNA sequence (GenBank accession number KP336491) 98% identical to that of several species of the genus Achromobacter. This is one of the first studies detailing the ability of Achromobacter sp. to utilize 2,2-DCP as the sole source of carbon and energy

In silico characterization of a novel dehalogenase (DehHX) from the halophile Pseudomonas halophila HX isolated from Tuz Gölü Lake, Turkey: insights into a hypersaline-adapted dehalogenase

Halogenated compounds represent potential long-term threats to human well-being and health and, therefore, the quest for microorganisms capable of degrading these hazardous substances merits urgent consideration. We have isolated a novel dehalogenase-producing bacterium from the hypersaline environment of Tuz Gölü Lake, Turkey and subsequently identified this isolate as Pseudomonas halophila HX. Under optimal culture conditions (pH 8.0, 15% NaCl, 30 °C, 200 rpm, 96 h culture time), the strain almost completely degraded (99.3%) 2,2-dichloropropionic acid (20 mM). The dehalogenase gene (dehHX) of the bacterium was amplified by PCR, and the deduced amino acid sequence of the DehHX was found to belong to a Group I dehalogenase and to share an 82% sequence identity to the dehalogenase DehI of Pseudomonas putida strain PP3. Interestingly, the pI of DehHX was more acidic (pI 3.89) than those of the non-halophilic dehalogenases (average measured pI 5.95). Homology-based structural modeling revealed that the surface of DehHX was unusually negatively charged due to the higher presence of acidic residues, which accounts for the uncommonly low pI seen in DehHX and explains the mechanism of adaptation that contributes to the exceptional halotolerance of the enzyme. The excess surface acidic residues were beneficial in enhancing the water-binding capacity, a crucial feature for preserving the stability and solubility of DehHX in highly saline conditions. In summary, we suggest that bio-prospecting for halogenated compound-degrading microorganisms in highly saline environments is a practical and safe strategy for the bioremediation of contaminated coastal areas

Isolation and identification of bacteria degrading 2,2-dichloropropionic acid in water sample

The widespread use of herbicides containing 2,2-dichloropropionic acid (2,2-DCP) as a recalcitrant halogen compound poses significant environmental risks and can be harmful for human. Consequently, it is important that the bio-based detoxification method is developed in an environmental manner. This study was aimed to isolate and identify a possible degradation 2,2-DCP bacterial strain as the sole source of carbon. A bacterial dehalogenase producing 2,2-DCP was isolated named as WM. The WM strain was shown to have 98% sequence identity and characteristics similar to Enterobacter sp. based on 16s rRNA analysis, biochemical and morphological tests. Phylogenics analysis showed that the WM strain is Enterobacter sp.. In media with 20 mM 3CP, the bacteria were well growing at 37°C, although an optimal chloride ion release was 0.48 μmol Cl/mL. Our finding is first report of an Enterobacter sp. strain which can use 2, 2-DCP as sole carbon source in a competent manner

In silico and empirical approaches toward understanding the structural adaptation of the alkaline-stable lipase KV1 from Acinetobacter haemolyticus

Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity