Molecular Cloning and Expression of Haloacid Dehalogenase Gene from a Local Pseudomonas aeruginosa ITB1 Strain and Tertiary Structure Prediction of the Produced Enzyme

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.

Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1

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

Utilisation of 2,2DCP by Staphyloccocus aureus ZT and In Silico Analysis of Putative Dehalogenase

Halogenated compound such as 2,2-dichloropropionic acid is known for its toxicity and polluted many areas especially with agricultural activities. This study focused on the isolation and characterization of the bacterium that can utilise 2,2-dichloropropionic acid from palm oil plantation in Lenga, Johor and in silico analysis of putative dehalogenase obtained from NCBI database of the same genus and species. The bacterium was isolated using an enrichment culture media supplemented with 20 mM 2,2-dicholoropropionic acid as a carbon source. The cells were grown at 30ËšC with cells doubling time of 2.00±0.005 hours with the maximum growth at A680nm of 1.047 overnight. The partial biochemical tests and morphological examination concluded that the bacterium belongs to the genus Staphylococcus sp.. This is the first reported studies of  Staphylococcus sp. with the ability to grow on 2,2-dichloropropionic acid. The genomic DNA from NCBI database of the same species was analysed assuming the same genus and has identical genomic sequence. The full genome of Staphylococcus sp. was screened for dehalogenase gene and  haloacid dehalogenase gene was detected in the mobile genetic element of the species revealed that the dehalogenase sequence has little identities to the previously reported dehalogenases.The main outcome of the studies suggesting an in situ bioremediation can be regarded as a natural process to detoxify the contaminated sites provided that the microorganisms contained a specialised gene sequence within its genome that served the nature for many long years. Whether microorganisms will be successful in destroying man-made contaminants entirely rely on what types of organisms play a role in in situ bioremediation and which contaminants are most susceptible to bioremediation.Â

Alternative Bioremediation Agents against Haloacids, Haloacetates and Chlorpyrifos Using Novel Halogen-Degrading Bacterial Isolates from the Hypersaline Lake Tuz

The indiscriminate use of chemical pesticides alongside the expansion of large-scale industries globally can critically jeopardize marine ecology and the well-being of mankind. This is because the agricultural runoffs and industrial effluents eventually enter waterways before flowing into highly saline environments i.e., oceans. Herein, the study assessed two novel bacterial isolates, Bacillus subtilis strain H1 and Bacillus thuringiensis strain H2 from the hypersaline Lake Tuz in Turkey to degrade recalcitrant haloalkanoic acids, haloacetates and chlorpyrifos, and consequently, identify their optimal pollutant concentrations, pH and temperature alongside salt-tolerance thresholds. Bacillus strains H1 and H2 optimally degraded 2,2-dichloropropionic acid (2,2-DCP) under similar incubation conditions (pH 8.0, 30 °C), except the latter preferred a higher concentration of pollutants as well as salinity at 30 mM and 35%, respectively, while strain H1 grew well on 20 mM at <30%. While both isolates could degrade all substrates used, the dehalogenase gene from strain H1 could not be amplified. Capacity of the H2 bacterial isolate to degrade 2,2-DCP was affirmed by the detection of the 795 bp putative halotolerant dehalogenase gene after a successful polymerase chain reaction (PCR) amplification. Hence, the findings envisage the potential of both isolates as bio-degraders of recalcitrant halogenated compounds and those of the same chemical family as chlorpyrifos, in saline environments.

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 molCl–/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.

Insights into origins and function of the unexplored majority of the reductive dehalogenase gene family as a result of genome assembly and ortholog group classification

Classifying all reductive dehalogenase genes from organohalide respiring bacteria, including nine newly closed genomes, predicts function and conserved synteny within species.

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.