Aims: A 2,2-dichloropropionic acid (2,2-DCP) naturally degrading bacterial species, strain SN1 was successfully isolated from cow dung capable of utilizing the substance as the sole carbon source and energy. Methodology and results: Strain SN1 was preferred over other strains (SN2, SN3 and SN4) following observations on its rapid growth in 20 mM 2,2-DCP liquid minimal media. Since strain SN1 clearly exhibited tolerance towards 2,2-DCP, its growth in various concentrations (10 mM, 20 mM, 30 mM and 40 mM) of the substance was evaluated. The study found the bacteria grew particularly well in 20 mM 2,2-DCP with the highest chloride release of 39.5 µmole Cl- /mL while exhibiting a remarkably short doubling time of 3.85 h. In view of such notable characteristics, species identification via Biolog GEN III system and 16S rRNA analysis was performed and established strain SN1 as Bacillus cereus. Conclusion, significance and impact of study: Considering the rapid growth of B. cereus strain SN1 in such medium, its employment as a bioremediation agent to treat 2,2-DCP contaminated soils may prove beneficial. Moreover, this is the first reported case of a Bacillus sp. isolated from cow dung capable of utilizing 2,2-DCP. Therefore, further assessment into its ability to degrade other types of haloalkanoic acids merit special consideration.
Bacillus cereus

Aims: This study aims to describe the biochemical and kinetic properties of a dehalogenase produced by a bacterium, Bacillus cereus WH2 (KU721999), that is uniquely adept at degrading a β-haloalkanoic acid, i.e., 3-chloropropionic acid (3-CP), and using it as the bacterium’s sole carbon source. The bacterium was isolated from abandoned agricultural land in Universiti Teknologi Malaysia that was previously exposed to herbicides and pesticides. Methodology and results: The B. cereus impressively removed 97% of 3-CP after 36 h of culturing. The intracellular WH2 dehalogenase of the bacterium was purified 2.5-fold and has an estimated molecular mass of 37 kDa. The highest activity of the dehalogenase was achieved under conditions of 30 °C and pH 7. The metal ions Hg2+ and Ag2+ substantially repressed the enzyme’s activity, but the enzyme’s activity was uninhibited by dithiothreitol (DTT) and EDTA. The WH2 dehalogenase showed a higher affinity for 3-CP (Km = 0.32 mM, kcat = 5.74 s-1 ) than for 3-chlorobutyric acid (3-CB) (Km = 0.52 mM; kcat = 5.60 s-1 ). The enzyme was ~1.6-fold more catalytically efficient (kcat/Km) in dehalogenating the three-carbon substrate 3-CP (17.8 mM-1 s -1 ) than the four-carbon 3-CB (11.2 mM-1 s -1 ). Conclusion, significance and impact of study: The novel B. cereus bacterium isolated in this study may prove applicable as a bioremediation agent to cleaning environments that are polluted with β-halogenated compounds. Furthermore, such an approach to treat polluted environments is more sustainable and potentially safer than chemical treatments.

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 Cα 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.

Aims: Bioprospecting for lipases remains limited despite its great deal of industrial application. This study reports on the purification and characterization of a novel lipase KV1 from Acinetobacter haemolyticus strain KV1. Methodology and results: Strain KV1 was identified as A. haemolyticus using the 16S rDNA sequencing, phylogenetic and BIOLOG assessments. The intracellular lipase was purified to homogeneity using consecutive treatments of ammonium sulfate precipitation, dialysis and DEAE-cellulose ion exchange chromatography, affording ~3.5-fold of the purified lipase with an estimated relative molecular mass of 37 kDa. The PCR product of lipase KV1 revealed that the retrieved sequence contained the proposed complete lipase gene sequence at nucleic acid positions 1-954. The purified lipase exhibited its maximum relative activity at 40 °C and pH 8.0, respectively. Interestingly, the novel alkalophilic lipase KV1 retained its relative activities (> 50%) even up to 24 h between pH 7-11. Conclusion, significance and impact of study The findings revealed that relative activities of the intracellular lipase KV1 were the highest at 40 °C and pH 8.0, respectively. Pertinently, the remarkable stability of the lipase KV1 over a broad range of pH values (pH 7-11), as well as an optimum activity at 40 °C indicated it was an excellent enzyme for producing a wide range of industrial detergents, cleaning up enviro-agro-industrial wastes as well as catalysts in synthetic manufacturing processes. Therefore, its full characterization reported here deserves scientific and economic considerations.

Aims: This study was aimed to characterize a dehalogenase derived from Bacillus cereus SN1 isolated from cow dung. Methodology and results: Cell-free extract of Bacillus cereus SN1 was purified using ion exchange and gel filtration chromatography. Fraction B2 of gel filtration gave the highest enzyme specific activity (0.155 μmol CI¯/min/mg). The results of SDS-PAGE showed the enzyme was 25 kDa in size. The enzyme reached its optimum activity at 30 °C at pH 6, and was inhibited by Mercury(II) sulfate (HgSO4). The Km and kcat values were 0.2 mM and 1.22/sec, respectively. The partial dehalogenase gene sequence was amplified using Group I dehalogenase primers. The amplified gene sequence was designated as DehSN1. Conclusion, significance and impact of study Dehalogenase from Bacillus cereus strain SN1 revealed new characteristics of dehalogenase protein. The findings indicated that the DehSN1 dehalogenase is a promising candidate for further studies as a bioremediation agent for agricultural applications.

Aims: The surplus use of herbicide Dalapon® contains 2,2-dichloropropionic acid (2,2-DCP) poses great danger to human and ecosystem due to its toxicity. Hence, this study focused on the isolation and characterization of a dehalogenase producing bacteria from Sungai Skudai, Johor, capable of utilizing 2,2-DCP as a carbon source and in silico analysis of its putative dehalogenase. Methodology and results: Isolation of the target bacteria was done by using 2,2-DCP-enriched culture as the sole carbon source that allows a bacterium to grow in 20 mM of 2,2-DCP at 30 °C with the corresponding doubling time of 8.89 ± 0.03 h. The isolated bacterium was then designated as Klebsiella pneumoniae strain YZ based on biochemical tests and basic morphological examination. The full genome of K. pneumoniae strain KLPN_25 (accession number: RRE04903) which obtained from NCBI database was screened for the presence of dehalogenase gene, assuming both strains YZ and KLPN_25 were the same organisms. A putative dehalogenase gene was then identified as type II dehalogenase from the genome sequence of strain KLPN_25. The protein structure of the type II dehalogenase of KLPN_25 strain was then pairwise aligned with the crystal structure of L-2-haloacid dehalogenase (L-DEX) Pseudomonas sp. strain YL as the template, revealing the existence of conserved amino acids residues, uniquely known to participate in the dehalogenation mechanism. The finding thus implies that the amino acid residues of type II dehalogenase possibly shares similar catalytic functions with the L-DEX. Conclusion, significance and impact of the study In conclusion, this study confirmed the presence of new dehalogenase from the genus Klebsiella with potential to degrade 2,2-DCP from the river water. The structural information of type II dehalogenase provides insights for future work in designing haloacid dehalogenases.
Klebsiella pneumoniae--isolation & ; purification ; Computer Simulation ; Molecular Dynamics Simulation

Halogenated compounds create the most important class of xenobiotic which commonly lead to pollution. Some of these compounds are very toxic and cause enormous problems to human health and to the environment. Many of these toxic chemicals have been shown to occur in various extreme habitats. Pollutant-degrading microorganisms, adapted to grow in various environments, play an important role in the biological treatment of polluted extreme habitats. The presence of dehalogenase producing microorganisms in extreme habitat in particular is necessary since the enzyme can catalyze the removal of a halogen atom from a substrate. Therefore, it can reduce the toxicity of the halogenated compound and some are of interest for study in industrial application. Thermophiles, psychrophiles, acidophiles, alkaliphiles and halophiles are types of extremophiles. Knowledge of the biodegradation of toxic chemicals in extreme environment is limited. Here, examples of dehalogenase producing bacteria isolated from various extreme conditions and its special characteristics/features will be discussed in this review.