Characterization of dehalogenase for the degradation of 3-chloropropionic acid

The use of herbicides that contain halogenated compounds, for example 3-chloropropionic acid (3-CP) poses significant environmental hazards as well as detrimental to human. The research detailed here investigated the isolation and identification of bacteria strains that could degrade 3-CP as its sol...

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Main Author:	Muslem, Wafaa Hassan
Format:	Thesis
Language:	English
Published:	2017
Subjects:	QH Natural history
Online Access:	http://eprints.utm.my/id/eprint/79571/1/WafaaHassanMuslemPFBME2017.pdf
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spelling	my-utm-ep.795712018-10-31T13:00:04Z Characterization of dehalogenase for the degradation of 3-chloropropionic acid 2017 Muslem, Wafaa Hassan QH Natural history The use of herbicides that contain halogenated compounds, for example 3-chloropropionic acid (3-CP) poses significant environmental hazards as well as detrimental to human. The research detailed here investigated the isolation and identification of bacteria strains that could degrade 3-CP as its sole carbon source. Dehalogenase that can degrade 3-CP is rare in nature. In this study, two strains of dehalogenase producing bacteria capable of utilizing 3-CP were successfully isolated from abandoned agricultural land in Universiti Teknologi Malaysia. These bacteria were characterized by using 16S rRNA as well as biochemical analysis. Strain WH1 showed a 98 % sequence identity to Burkholderia cepacia with (accession number KU318403) whereas strain WH2 showed a 99% sequence identity to Bacillus cereus with (accession number KU721999). The results have shown that these bacteria were capable to grow in liquid minimal media supplied with 10 mM 3-CP as sole carbon source with doubling time of 43.62 h for WH1 and 14.75 h for WH2. Utilization of 3-CP was confirmed by detection of chloride ion released using halide ion assay technique for both strains which indicate their ability to degrade 3-CP. For further confirmation of 3-CP consumption, analysis by high performance liquid chromatography (HPLC) revealed that both B. cepacia WH1 and B. cereus WH2 effectively utilized ~100% of 10 mM 3-CP. This is the first report detailing both strains able to competently utilize 3-CP as their sole carbon source. Cell free extract of B. cereus strain WH2 was further characterized due to its faster growth on 3-CP compared to B. cepacia strain WH1. The intracellular dehalogenase from B. cereus WH2 was purified to homogeneity to afford a 2.5-fold (50 % yield) concentration with an estimated molecular mass of 37 kDa by SDS-PAGE analysis. Its highest enzyme activity was achieved at conditions of 30 oC and pH 7. While the activity of WH2 dehalogenase was substantially repressed by both Hg2+ and Ag2+, the enzyme was not inhibited by DTT and EDTA. Pertinently, kinetics evaluation revealed a higher affinity of the WH2 dehalogenase towards 3-CP than 3-chlorobutyric acid (3-CB), affording Km values of 0.32 mM (kcat 3.97 s-1) and 0.52 mM (kcat 4.35 s-1), respectively. The WH2 dehalogenase was ~1.6-fold catalytically more efficient (kcat/Km) in dehalogenating the three-carbon, 3-CP (12.4 mM-1 s-1) over the four-carbon, 3-CB (8.27 mM-1 s-1). From the data, it was identified that 3-CP degradation was not stimulated by co-factors, such as NAD+, NADH, NADP+, NADPH, FAD and CoA that did not affect the enzyme activity by demonstrating activities of <0.1 unite (g protein)-1. The amplified dehalogenase gene fragment was designated “deh-wh2” and subsequent analysis showed it belongs to Group II dehalogenase. Eight conserved residues that line the active site were identified: Asp10, Thr14, Ser117, Lys150, Tyr156, Ser174, Asn176 and Asp179. These residues are consistent with the residues found in the active site of DhlB, DehIVa and L-DEX. The product of 3-CP degradation was 3-hydroxypropionic acid based on HPLC. In conclusion, this study confirmed the presence of new dehalogenase isolated from various bacteria that have potential to utilize 3-CP, especially from contaminated environment. 2017 Thesis http://eprints.utm.my/id/eprint/79571/ http://eprints.utm.my/id/eprint/79571/1/WafaaHassanMuslemPFBME2017.pdf application/pdf en public phd doctoral Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering Faculty of Biosciences and Medical Engineering
institution	Universiti Teknologi Malaysia
collection	UTM Institutional Repository
language	English
topic	QH Natural history
spellingShingle	QH Natural history Muslem, Wafaa Hassan Characterization of dehalogenase for the degradation of 3-chloropropionic acid
description	The use of herbicides that contain halogenated compounds, for example 3-chloropropionic acid (3-CP) poses significant environmental hazards as well as detrimental to human. The research detailed here investigated the isolation and identification of bacteria strains that could degrade 3-CP as its sole carbon source. Dehalogenase that can degrade 3-CP is rare in nature. In this study, two strains of dehalogenase producing bacteria capable of utilizing 3-CP were successfully isolated from abandoned agricultural land in Universiti Teknologi Malaysia. These bacteria were characterized by using 16S rRNA as well as biochemical analysis. Strain WH1 showed a 98 % sequence identity to Burkholderia cepacia with (accession number KU318403) whereas strain WH2 showed a 99% sequence identity to Bacillus cereus with (accession number KU721999). The results have shown that these bacteria were capable to grow in liquid minimal media supplied with 10 mM 3-CP as sole carbon source with doubling time of 43.62 h for WH1 and 14.75 h for WH2. Utilization of 3-CP was confirmed by detection of chloride ion released using halide ion assay technique for both strains which indicate their ability to degrade 3-CP. For further confirmation of 3-CP consumption, analysis by high performance liquid chromatography (HPLC) revealed that both B. cepacia WH1 and B. cereus WH2 effectively utilized ~100% of 10 mM 3-CP. This is the first report detailing both strains able to competently utilize 3-CP as their sole carbon source. Cell free extract of B. cereus strain WH2 was further characterized due to its faster growth on 3-CP compared to B. cepacia strain WH1. The intracellular dehalogenase from B. cereus WH2 was purified to homogeneity to afford a 2.5-fold (50 % yield) concentration with an estimated molecular mass of 37 kDa by SDS-PAGE analysis. Its highest enzyme activity was achieved at conditions of 30 oC and pH 7. While the activity of WH2 dehalogenase was substantially repressed by both Hg2+ and Ag2+, the enzyme was not inhibited by DTT and EDTA. Pertinently, kinetics evaluation revealed a higher affinity of the WH2 dehalogenase towards 3-CP than 3-chlorobutyric acid (3-CB), affording Km values of 0.32 mM (kcat 3.97 s-1) and 0.52 mM (kcat 4.35 s-1), respectively. The WH2 dehalogenase was ~1.6-fold catalytically more efficient (kcat/Km) in dehalogenating the three-carbon, 3-CP (12.4 mM-1 s-1) over the four-carbon, 3-CB (8.27 mM-1 s-1). From the data, it was identified that 3-CP degradation was not stimulated by co-factors, such as NAD+, NADH, NADP+, NADPH, FAD and CoA that did not affect the enzyme activity by demonstrating activities of <0.1 unite (g protein)-1. The amplified dehalogenase gene fragment was designated “deh-wh2” and subsequent analysis showed it belongs to Group II dehalogenase. Eight conserved residues that line the active site were identified: Asp10, Thr14, Ser117, Lys150, Tyr156, Ser174, Asn176 and Asp179. These residues are consistent with the residues found in the active site of DhlB, DehIVa and L-DEX. The product of 3-CP degradation was 3-hydroxypropionic acid based on HPLC. In conclusion, this study confirmed the presence of new dehalogenase isolated from various bacteria that have potential to utilize 3-CP, especially from contaminated environment.
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Muslem, Wafaa Hassan
author_facet	Muslem, Wafaa Hassan
author_sort	Muslem, Wafaa Hassan
title	Characterization of dehalogenase for the degradation of 3-chloropropionic acid
title_short	Characterization of dehalogenase for the degradation of 3-chloropropionic acid
title_full	Characterization of dehalogenase for the degradation of 3-chloropropionic acid
title_fullStr	Characterization of dehalogenase for the degradation of 3-chloropropionic acid
title_full_unstemmed	Characterization of dehalogenase for the degradation of 3-chloropropionic acid
title_sort	characterization of dehalogenase for the degradation of 3-chloropropionic acid
granting_institution	Universiti Teknologi Malaysia, Faculty of Biosciences and Medical Engineering
granting_department	Faculty of Biosciences and Medical Engineering
publishDate	2017
url	http://eprints.utm.my/id/eprint/79571/1/WafaaHassanMuslemPFBME2017.pdf
_version_	1747818258927452160

Characterization of dehalogenase for the degradation of 3-chloropropionic acid

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