scholarly journals Cloning of Haloacid Dehalogenase Gene from Bacillus Cereus Local Strain with the Addition of Restriction Sites

2015 ◽  
Vol 16 ◽  
pp. 314-320
Author(s):  
Idris ◽  
Enny Ratnaningsih
Keyword(s):  
Bacillus Cereus ◽  
Local Strain ◽  
Haloacid Dehalogenase ◽  
Restriction Sites ◽  
Dehalogenase Gene

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

2021 ◽  
Vol 10 (4) ◽  
pp. 857-863
Author(s):  
Enny Ratnaningsih ◽  
Rachmad Ade ◽  
Rindia Maharani Putri ◽  
Idris Idris
Keyword(s):  
Bacillus Cereus ◽  
Chloride Ion ◽  
Local Strain ◽  
Luria Bertani ◽  
Chloride Ions ◽  
Monochloroacetic Acid ◽  
Iptg Induction ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Dehalogenase Gene

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

scholarly journals Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

2018 ◽  
Vol 22 (2) ◽  
pp. 55
Author(s):  
Enny Ratnaningsih ◽  
Idris Idris
Keyword(s):  
Recombinant Protein ◽  
Bacillus Cereus ◽  
Tertiary Structure ◽  
Specific Activity ◽  
Halogen Bond ◽  
Expression System ◽  
Industrial Sector ◽  
Cloning And Expression ◽  
Haloacid Dehalogenase ◽  
Organohalogen Compounds

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

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

REAKTOR ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 59-64
Author(s):  
Enny Ratnaningsih ◽  
Sulistiya Nirta Sunaryo ◽  
Idris Idris ◽  
Rindia Maharani Putri
Keyword(s):  
Bacillus Cereus ◽  
Recombinant Expression ◽  
Host Cells ◽  
Monochloroacetic Acid ◽  
Affinity Column ◽  
One Pot ◽  
Crude Enzyme Extract ◽  
Local Strains ◽  
Haloacid Dehalogenase ◽  
Purification System

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

Cloning and Sequence Analysis of a Plasmid-encoded 2-Haloacid Dehalogenase Gene fromPseudomonas putidaNo. 109

1994 ◽  
Vol 58 (1) ◽  
pp. 160-163 ◽  
Author(s):  
Haruhiko Kawasaki ◽  
Tohru Toyama ◽  
Takuya Maeda ◽  
Humiko Nishino ◽  
Kenzo Tonomura
Keyword(s):  
Sequence Analysis ◽  
Haloacid Dehalogenase ◽  
Dehalogenase Gene

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

2021 ◽  
Vol 24 (5) ◽  
pp. 161-169
Author(s):  
Enny Ratnaningsih ◽  
Lousiana Dwinta Utami ◽  
Nurlaida Nurlaida ◽  
Rindia Maharani Putri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Tertiary Structure ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Local Strains ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Cloned Gene ◽  
Dehalogenase Gene

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.

scholarly journals Expression of haloacid dehalogenase gene and its molecular protein characterization from Klebsiella pneumoniae ITB1

2018 ◽  
Vol 22 (1) ◽  
pp. 1
Author(s):  
Ridani Rino Anggoro ◽  
Enny Ratnaningsih
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Sites ◽  
Flame Retardants ◽  
Specific Activity ◽  
Expression System ◽  
Three Dimensional ◽  
Protein Characterization ◽  
Dimensional Structure ◽  
Haloacid Dehalogenase ◽  
Dehalogenase Gene

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.

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