Haloacid dehalogenases of Rhizobium sp. and related enzymes: Catalytic properties and mechanistic analysis

Adamu, Aliyu; Wahab, Roswanira Abdul; Aliyu, Firdausi; Aminu, Aminu Hamza; Hamza, Mariya Musa; Huyop, Fahrul

doi:10.1016/j.procbio.2020.02.002

Cited by 17 publications

(15 citation statements)

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“…The 2-haloacid dehalogenases have been classified according to amino acid sequence conservation and substrate selectivity (Wang et al, 2018;Adamu et al, 2020). These enzymes are classified into four types according to their substrate specificities and product configurations: D-2-haloacid dehalogenase (D-DEX, EC 3.8.1.9), L-2-haloacid dehalogenase (L-DEX, EC 3.8.1.2), configuration-inverting DL-2-haloacid dehalogenase (DL-DEXi, EC 3.8.1.10), and configuration-retaining DL-2haloacid dehalogenase EC 3.8.1.11) (Zakary et al, 2021).…”

Section: Isolation Sources and Classification Of 2-haloacid Dehalogenasesmentioning

confidence: 99%

“…Microorganisms growing in environments polluted by organic halogenated compounds have the potential to transform these compounds owing to the presence of enzymes that catalyze dehalogenation in their cells, called dehalogenases (Atashgahi et al, 2018;Oyewusi et al, 2020bOyewusi et al, , 2021b. Among them, 2-haloacid dehalogenases are a family of critical enzymes that hydrolytically catalyze the dehalogenation of 2-haloacids to form corresponding 2hydroxy acids (Kurihara and Esaki, 2008;Adamu et al, 2020). They cannot only degrade toxic pollutants with low energy consumption but also have a wide substrate profile and high catalytic efficiency.…”

Section: Introductionmentioning

confidence: 99%

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Mini Review: Advances in 2-Haloacid Dehalogenases

et al. 2021

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The 2-haloacid dehalogenases (EC 3.8.1.X) are industrially important enzymes that catalyze the cleavage of carbon–halogen bonds in 2-haloalkanoic acids, releasing halogen ions and producing corresponding 2-hydroxyl acids. These enzymes are of particular interest in environmental remediation and environmentally friendly synthesis of optically pure chiral compounds due to their ability to degrade a wide range of halogenated compounds with astonishing efficiency for enantiomer resolution. The 2-haloacid dehalogenases have been extensively studied with regard to their biochemical characterization, protein crystal structures, and catalytic mechanisms. This paper comprehensively reviews the source of isolation, classification, protein structures, reaction mechanisms, biochemical properties, and application of 2-haloacid dehalogenases; current trends and avenues for further development have also been included.

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Section: Isolation Sources and Classification Of 2-haloacid Dehalogenasesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mini Review: Advances in 2-Haloacid Dehalogenases

et al. 2021

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“…The activity of haloacid dehalogenase bcfd1 was quantitatively analyzed by measuring the amount of ( free chloride ions into the medium as a result of MCA degradation by haloacid dehalogenase (Adamu, et al, 2020;Torz and Beschkov, 2005;Ang, et al, 2018). The measurement of chloride ions in solution was carried out using the Bergmann and Sanik method (Bergmann and Sanik, 1957).…”

Section: Enhanced Activity Of Haloacid Dehalogenase Upon One-pot Purificationmentioning

confidence: 99%

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

Ratnaningsih

Sunaryo²,

Idris³

et al. 2021

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In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

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“…YL listed Asp, Thr, Arg, Ser, Lys, Tyr, and Asn on its active site [ 14 ] and haloacid dehalogenase from S. tokodaii that identified Asp, Thr, Arg, Lys, Tyr, and Ser as its essential catalytic residues [20]. It is well known that Asp is the key catalytic residue that is conserved in most L -2 -haloacid dehalogenase [21,22,23 ] as this residue plays an essential role in nucleophilic attack to the substrate.…”

Section: Prediction Of Catalytic Residuesmentioning

confidence: 99%

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

et al. 2021

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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.

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Haloacid dehalogenases of Rhizobium sp. and related enzymes: Catalytic properties and mechanistic analysis

Cited by 17 publications

References 44 publications

Mini Review: Advances in 2-Haloacid Dehalogenases

Mini Review: Advances in 2-Haloacid Dehalogenases

Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

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

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